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Protein Folding and Expression

Folding, Expression and Analysis

Clontech, A Takara Bio Inc. Company • www.clontech.com/takara2

1. Introduction......................................................................................................... 3

2. ExpressionSystems............................................................................................... 4Human Cell-Free Expression System ......................................................................................................................................4Brevibacillus Expression System II ...........................................................................................................................................6B. subtilis Secretory Protein Expression System II ..............................................................................................................8

3. ProductsforIncreasedProteinYieldandPurity.................................................... 9pCold Expression Vectors ............................................................................................................................................................9pCold TF Vector ........................................................................................................................................................................... 10pCold™ ProS2 DNA ..................................................................................................................................................................... 10mRNA Interferase™-MazF Enzyme ........................................................................................................................................ 10SPP System .................................................................................................................................................................................... 11

4. MammalianExpressionVectors............................................................................12pBApo-CMV Vectors ................................................................................................................................................................... 12pBApo-EF1a .................................................................................................................................................................................. 12pDON-AI-2 ...................................................................................................................................................................................... 13pMEI-5.............................................................................................................................................................................................. 13pDON-5 ........................................................................................................................................................................................... 14

5. Folding................................................................................................................15Chaperone Plasmid Set ............................................................................................................................................................. 15Chaperonin GroE ......................................................................................................................................................................... 16Corystein™ (Purothionin) Reagent ........................................................................................................................................ 16Refolding CA Kit ........................................................................................................................................................................... 16

6. ApplicationNotes................................................................................................17High-level Secretion of Recombinant Protein using the Brevibacillus Expression System ............................. 17The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coli ....................................... 20Unfolding the Potential of Proteins ..................................................................................................................................... 22

7. FAQs....................................................................................................................24SPP System™ (Single Protein Production System) .......................................................................................................... 24pCold Expression Vectors ......................................................................................................................................................... 25Refolding CA Kit ........................................................................................................................................................................... 26Chaperone Plasmid Set ............................................................................................................................................................. 27

8. ProteinSequencingandAnalysisProducts...........................................................28

9. HighFidelityPCREnzymes...................................................................................29

10.TakaraRelatedProducts......................................................................................30

11.ClontechRelatedProducts...................................................................................31

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Contents

Clontech, A Takara Bio Inc. Company • www.clontech.com/takara 3

1

Refolding CA KitOptimize Refolding of

Inclusion Bodies

Corystein™ Reagent Reforming Disul�de Bonds

Chaperone Plasmid SetMaximize Soluble Active Protein

Chaperonin GroE Increase Level of Active Protein

Chaperone AssistedFolding

Insoluble Protein or Protein Toxic to Cell

Protein Folding Products

Takara’s Protein Products

Active Protein Ready for puri�cation and/or analysis

Protein Expression Products

pCold TF DNA Increase Expression using Trigger Factor

pCold Pro S2Express Soluble Fusion Proteins

pCold VectorsIncrease Recombinant Protein Yield

Increase ProteinYield and Purity

MammalianExpression

Brevibacillus Expression System IIHigh E�ciency Protein Production

High YieldActive Protein

SPP System™ KitsSingle Protein Production System

mRNA Interferase™ PlasmidMazF Enzyme

pBApo-EF1α Vectors Strong promoter for increased expression

pDON-AI-2 Vectors Retroviral Vector for High-e�ciency

Gene Transduction

pMEI-5 Vectors Retroviral Vector for High Expression

pDON-5 Vector Retroviral Vector for High-e�ciency

Gene Transduction and High Expression

pBApo-CMV Vectors All-purpose Gene Expression Vectors

Human Cell Free Expression SystemHigh E�ciency Cell-free Protein Production

B. subtilus Expression SystemSecretory Protein Production

Protein expression is not an easy process; however, it can be made easier with the help of products that aid expression and folding. There are many factors which can prohibit the production of functional protein. Often it is not possible to predict which factors may impact expression of a particular protein of interest. In order for production of your target protein to be successful, map the process out from the beginning. The expression process consists of four steps: cloning, expression, purification, and characterization. Each step can be optimized to obtain a sufficient amount of functional protein. Consider the end use of the protein. Is it for biochemical studies or for structural analysis (NMR, X-ray crystallography)? The answer should help determine the best production process for your needs. Takara Bio offers a wide selection of tools to aid every step of the protein production process.

Intro

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Introduction


HumanCell-FreeExpressionSystem

Features• Easy-to-usesystemallowsgenerationofproteininaslittleas1hinasingle-tubereaction• Provideshigheryieldoffunctionalproteinandgreaterconsistencythanrabbit

reticulocyteorwheatgermin vitrotranslationsystems• Excellentwithchallengingproteinssuchaslargeproteins(over150kDa)andproteins

requiringpost-translationalmodification• Amenabletohigh-throughputscreening;bulksizesavailable.

Applications• Expressionoftoxicproteinsthatarelethaltohostcellsofin vivoexpressionsystems• Rapidanalysisofproteinfunction• Rapidanalysisofmutationseriesortruncationseries:generateproteinquicklyand

assessfunctionalityusingyourdownstreamassay• High-throughputproteomicstudies• Expressionofproteinsthatareeasilydegradedorinsolubleinconventionalin vivo

expressionsystemssuchasE. coli

DescriptionTheHumanCell-FreeProteinExpressionSystemfromTakaraBioiseasytouse.Thesingle-tubereactioniseasilyassembledandproteinsynthesisiscompleteinaslittleas1hat32°C.TheHumanCell-FreeProteinExpressionSystemprovideshighyield(e.g.,50µg/mlofhumaneIF4G)offunctionalprotein,includingproteinsrequiringmodificationssuchasglycosylation,phosphorylation,orfattyacylation.Excellentyieldisobservedevenwithlargeproteins(over150kDa).GenesofinterestmaybeclonedrapidlyintopT7-IRESvectorsusingIn-Fusioncloningtechnology.Afterexpression,proteinswithN-terminalorC-terminalHis-tagorN-terminalMyctagcanbegenerated,dependingonchoiceofvector.Bulksizesareavailableforhigh-throughputstudies;[email protected].

In vitrotranslationhasmanyadvantagesforproteinexpression:itisexcellentforrapidstudiesofproteinfunctionorfeatures,amenabletohigh-throughputstudies,usefulforproteinsthataredegradedorinsolublewithin vivosystems,andcanbeusedtogeneratelethalproteinsthatcannotbeexpressedusingin vivosystemsduetotoxicity.Incontrasttoexpressionusingprokaryotichostcells,in vitrotranslationsystemsalsoallowpost-translationalmodificationssuchasglycosylation,phosphorylation,andfattyacylation.

Kit Components (for 10 × 20-μL reactions)(1)CellLysate*1 100μL(2)Mixture-1 60μL(3)Mixture-2*2 10μL(4)Mixture-3*2 20μL(5)T7RNAPolymerase(200U/μL) 10μL(6)pT7-IRESVector(0.5μg/μL) 20μL(7)ControlVector*3(0.3μg/μL) 5μL*1: Dissolvejustpriortouse;gentlyandthoroughlymixwithamicropipetteand

useimmediately.Afteruse,promptlystoreat–80°C.Note: Althoughfivecyclesoffreeze-thawgenerallywouldnotleadtoanydeclineinperformance,thecelllysateshouldbestoredinaliquotsoftherequiredvolume.

*2: Mixture-2andMixture-3containprotein.Toavoidproteindeactivation,donotstirexcessivelyorvortex.Mixture-2containsanHN-taggedprotein.

*3: Thisvectorharborsaβ-galactosidasegene.

pT7-IRES Vectors:Eachvectorprovides20µgDNAataconcentrationof0.5µg/µL(40µLtotalvolume).

pT7-IRES Vector InformationVectorsinthepT7-IRESseriesincludeaT7promoterandEMCVIRESsequencetofacilitatetranscriptionandtranslationintheHumanCell-FreeProteinExpressionSystem,aswellasaMultipleCloningSite(MCS),convenienttags(N-orC-terminalHis-tagorMyctagsequences),aFactorXaproteasecleavagesitefortagremoval,poly-Asignal,andT7terminator.

• pT7-IRESHis-NDNA(Cat.#3290)encodesanN-terminalHis-tag• pT7-IRESHis-CDNA(Cat.#3291)encodesaC-terminalHis-tag• pT7-IRESMyc-NDNA(Cat.#3292)encodesanN-terminalc-Myctag

Storage• HumanCell-FreeProteinExpressionSystem(Cat.#3281):–80°C• pT7-IRESVectors(Cat.#s3290,3291,3292):–20°C

Related In-Fusion Cloning ProductsForrapidandeasycloning,usetheIn-FusionHDCloningSystem(ClontechCat.#639645/639646/639692/639647)orIn-FusionHDCloningSystemCE(ClontechCat.#639636/639637/639693/639638)togeneratepT7-IRESconstructswithyourinsertofinterest.

Human Cell-Free Protein Expression System 3281 10 rxns pT7-IRES His-N DNA 3290 20 µg pT7-IRES His-C DNA 3291 20 µg pT7-IRES Myc-N DNA 3292 20 µg

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NoteThis product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc.Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC.If you require licenses for other use, please contact us by phone at +81 77 543 7247 or from our website at www.takara-bio.com.Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements.

Shipping at － 20℃Store at － 20℃

pT7-IRES His-N DNA

Code No. 3290Size: 20 μg

Lot No.Concentration : 0.5 μg/μlVolume : 40 μl

＊ 2 years from date of receipt under proper storage conditions.

Regarding protocol for protein expression, please refer to the product manual for Human Cell-Free Pro-tein Expression System (Cat. #3281).

Description :pT7-IRES DNA series are expression vectors designed for Human Cell-Free Protein Expression System. Tag sequence such as His-Tag or c-Myc Tag, Factor Xa cleavage site, Multiple Cloning Site (MCS), polyA signal, and T7 terminator are located at the downstream of T7 promoter and EMCV IRES. There are vectors with some arrangements for the kind of Tag and the location.pT7-IRES His-N DNA is an expression vector including His-Tag sequence. Using pT7-IRES His-N DNA together with Human Cell-Free Protein Expression System (Cat. #3281) enables the expression of your target protein as N-terminal fusion of His-Tag. Factor Xa cleavage site is inserted so that His-Tag can be removed from the expressed fusion protein.Target gene cloned into MCS in frame is transcribed as RNA-containing-EMCV IRES under the control of T7 promoter. By the effect of EMCV IRES, designed to promote protein translation initiation, efficient high level expression can be performed in Human Cell-Free Protein Expression System.

Form : 10 mM Tris-HCl, pH8.0 1 mM EDTA

Preparation : Purified by ion-exchange column.

Chain length : 3,249 bp

EMCVIRES

Ampr

ColE1 ori

His-TagFactor Xa Site

Multiple Cloning SitepolyA T7 terminator

pT7-IRES His-N DNA(3,429 bp)

PT7

Purity : 1． Confirmed to maintain the region from T7 promoter to T7 terminator

by dideoxy sequencing method.2． Shown to be cleaved at a single site by EcoR I and at two sites by

Hind III.

Usage : Protein expression using Human Cell-Free Protein Expression System.

Vector map for pT7-IRES His-N DNA :

MCS :Nco I

EMCV IRES Nhe I His-Tag Factor Xa Nde I Sac I Xho I5’- TAACGT・・・・・・TAATATGGCCACAACC ATG GC T AGC CAC CAT CAC CAT CAC CAT AT C GAA GGG CGC CAT AT G GAG CT C CT C GAG3’-AT TGCA・・・・・・AT TATACCGGTGT TGG TAC CGA T CG GTG GTA GTG GTA GTG GTA TAG C T T CCC GCG GTA TAC C T C GAG GAG C T C

Met Ala Ser His His His His His His Ile Glu Gly Arg His Met Glu Leu Leu Glu

Hinc IIBamH I EcoR I Spe I Sal I Pst I Xba I End

GGA T CC GAA T T C AC T AGT GT C GAC C TG CAG T C T AGA TAG GTAAT C-3’CC T AGG C T T AAG TGA T CA CAG C TG GAC GT C AGA T C T AT C CAT TAG-5’Gly Ser Glu Phe Thr Ser Val Asp Leu Gln Ser Arg

Figure 1. Map of pT7-IRES vector.


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120

100

80

60

40

20

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Relative absorbance

Time (hour)

Mixture-2(ー)Mixture-2(＋)

Figure 3. Time course of β-galactosidase expression and effect of translation enhancement factor.Eightreplicatesofβ-galactosidasein vitrotranslationwereperformedusing1µLofControlVectorperreaction.Ateachoftheindicatedtimepoints(0,0.5,1,2,3,4.5,6,and8hafterstartofthereaction),onereactiontubewasremovedandusedforβ-galactosidaseactivityassaywithO-nitrophenyl-β-D-galactopyranoside(ONPG)asthesubstrate.Aseparatesetofreactionswereconductedinabsenceofthetranslationenhancementfactor(Mixture-2).Theactivityofβ-galactosidaseincreasedovertimetopeakatapproximately4.5h.Additionally,thepresenceofMixture-2containingtranslationenhancementfactormarkedlyincreasedyieldofactiveprotein.

Figure 2. Principle of the Human Cell-Free Protein Expression System. A)Inaneasyandsimpleprotocol,targetproteintranslationisinitiatedbyaddingpT7-IRESVectorcontainingthetargetgenecassetteandotherkitcomponents.B)ThetargetgeneRNAtranscribedfromthepT7-IRESVectorhasanIRESsequencedesignedtopromoteproteintranslationinitiation.Asproteinsynthesisprogresses,thetranslationinitiationfactorfromthecelllysatebecomesinactivated.Thetranslationenhancementfactorinthereactionmixture,however,reactivatesthisinactivatedtranslationinitiationfactorandtherebymaintainsahighleveloftranslation.

Figure 4. Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System.In vitrotranslationreactionswereperformedtosynthesizehumanDicer(200kDa,lane2)orhumaneIF4G(170kDa,lane3)protein.ReactionswereanalyzedbySDS-PAGEandCoomassiebluestaining.Arrowheadsindicatetargetproteins.Lane1,negativecontrol.

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Features• Efficientproductionofsecretedorintracellulartargetproteins• Producesnegligibleamountsofextracellularprotease–productsremainintactin

culturemedium• UnlikeE.coli,producesnoendotoxins• Proteinsareproducedinactiveform• Easytoculture,handle,andsterilize

DescriptionBrevibacilluschoshinensisisagram-positivebacteriumwithexceptionalcapacityforheterologousproteinexpression.

TheBrevibacillusExpressionSystemIIenableshighlyefficientproductionoftargetproteininsecretedform.Thissystemallowshighyieldofactiveproteinsandiswell-suitedforexpressionofeukaryoticproteins.TheBrevibacillussystemisnearlyfreeofproteases,whichfacilitatesproductionofintactproteinproducts.

ExamplesofsuccessfullyexpressedproteinscanbeseeninTable1.Thisincludesexpressionofenzymes,antigens,andcytokines.Eachproteinwasproducedataveryhighlevelofexpressionandconfirmedtohavenativebiologicalactivity.Inaddition,proteinsfromtaxonomicallydistantorganismsweresuccessfullyproduced,suchaseubacteria,archaebacteria,eukaryotes,andviruses.

TheBrevibacillussystemfacilitatesdisulfidebondformation(commonlyrequiredinproteinsofeukaryoticorigin).Inaddition,B.choshinensisservesasanexcellenthostforintracellularproteinproduction,frequentlyproducingintracellularproteinsinsolubleforminthecytoplasmwithoutforminginclusionbodies.TheBrevibacillussystemoftenworksbetterthanE.coliforexpressionofparticulartargets.

UtilizingHis-tagcontainingvectors(pNC-HisE,pNC-HisF,pNC-HisT,pNI-His)allowseffectivepurificationoftheexpressedtargetprotein.Tagscanberemovedbyproteasetreatmentfollowingpurification.

BrevibacillusExpressionSystemII

Brevibacillus Expression System II HB200 1 Kit Brevibacillus choshinensis Competent Cells HB116 100 µL x 10 pNC-HisE DNA HB123 10 µg pNC-HisF DNA HB122 10 µg pNC-HisT DNA HB121 10 µg pNCMO2 DNA HB112 10 µg pNY326-BLA DNA HB114 1 µg pNI DNA HB131 10 µg pNI-His DNA HB132 10 µg pNY326 DNA HB111 10 µg

Table 1: Example of Proteins Expressed using the Brevibacillus Expression System

Proteins Origins Production（ g/L ）Enzymes

α-amylase B. licheniformis 3.7Sphingomyelinase B. cereus 3Xylanase B. halodurans 0.2CGTase B. macerans 1.5Chitosanase B. circulans 1.4Hyper thermo-stable protease A. pernix 0.1Hyper thermo-stable nuclease P. horikoshii 0.7PDI human 1AntigensSurface antigen E. rhusiopathiae 0.9Surface antigen T. pallidum 0.8Cytokin sEGF human 1.5IL-2 human 0.6NGF mouse 0.2IFN- γ chicken 0.5TNF-α bovine 0.4GM-CSF bovine 0.2GH flounder 0.2

Proteins Origins Production（ g/L ）

α-amylase B. licheniformis 3.7Sphingomyelinase B. cereus 3.0Xylanase B. halodurans 0.2CGTase B. macerans 1.5Chitosanase B. circulans 1.4Hyper thermo-stable protease A. pernix 0.1Hyper thermo-stable nuclease P. horikoshii 0.7PDI human 1.0

Surface antigen E. rhusiopathiae 0.9Surface antigen T. pallidum 0.8

eEGF human 1.5IL-2 human 0.6NGF mouse 0.2IFN- γ chicken 0.5TNF-α bovine 0.4GM-CSF bovine 0.2GH flounder 0.2

Product Name Catalog # Quan tyBrevibacillus Expression System II HB200 KitKit Components

Expression VectorspNY326A DNA HB111 10 µgpNCMO2 DNA HB112 10 µgControl VectorpNY326-BLA DNA HB114 1 µgCompetent CellsBrevibacillus choschinesis Competent cells HB116 100 µL x 10 tubesMT Medium 1 ml x 10Solu on A 1 mlSolu on B 1 ml x 2

System Components:

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Vector Name Vector Type Expression Vector Lac Operator His-Tag Sec Signal Peptide Construct in Protease cleavage site X-terminator

pNC-HisE(5,263bp) ShuttleVector Secretory Yes Yes Yes E. coli Enterokinase No

pNC-HisT(5,260bp) ShuttleVector Secretory Yes Yes Yes E. coli Thrombin No

pNC-HisF(5,260bp) ShuttleVector Secretory Yes Yes Yes E. coli FactorXa No

pNI-HisDNA(5,079bp) ShuttleVector Intracellular Yes Yes No E. coli Enterokinase No

pNIDNA(5,055bp) ShuttleVector Intracellular Yes No No E. coli No No

pNY326(3.4Kb) Expression Secretory No No Yes Brevibacillus No Yes

pNCMO2(5.2kb) ShuttleVector Secretory No No Yes E. coli No Yes

pNY326-BLA PositiveControl Secretory IncludesageneencodingBacillus licheniformisa-amylase(55kDa)

Vectors for the Brevibacillus System

AllshuttlevectorsbetweenB. choshinensisandE. colicontaintheP2promoter,whichisoneofthefivepromotersthatcontroltranscriptionofthecellwallproteingene(HWP).ThispromoterfunctionsonlyinB. choshinensis andnotinE. coli, therebyensuringrobustproteinproductiononlyin B. choshinensis.

ThepNY326 vector* ismaintainedmorestablythanpNCorpNIvectorsinthehostcellsduetomuchweakerpromoteractivityandsmallersize(3.4kb).Thehoststrainscontainingthevector canberepeatedlysubcultured,maybeusedforscaled-upproduction,andwillcontinuetostablyproduceprotein.ThepNY326vectormustbeconstructedbyaone-stepmethodusingB. choshinensis.BrevibacilluschoshinensisCompetentCellsareusedasthetransformationhost.

*CanonlybemaintainedinB. choshinensis

Nmrrep

ori –P5 promoter

sec signal peptide

multiple cloning site

X-terminator

ori +

pNY326(3.4 kp)

Amp r

ColE1 ori

Nm r

rep

ori ＋

ori –

pNCMO2 (5.2 kb）

P2 promoterLac operator

sec signal peptide

His-TagMCS

ori +

rep

NmR

AmpR

ColE1 ori

ori –

pNC-HisT(5,260 bp)


sec signal peptide

His-TagMCS

ori＋

rep

NmR

AmpR

ColE1 ori

ori－

pNC-HisF(5,260 bp)


sec signal peptide

His-TagMCS

ori＋

rep

NmR

AmpR

ColE1 ori

ori－

pNC-HisE(5,263 bp)

P2 promoter Lac operator

multi cloning site

ori +

rep

Nmr

Ampr

ColE1 ori

ori –

pNI-His DNA(5,079 bp)

His-TagP2 promoter

Lac operator

multi cloning site

ori +

rep

Nmr

Ampr

ColE1 ori

ori –

pNI DNA(5,055 bp)

Choosing a Brevibacillus Vector?

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Clontech offers a broad range of products for purifying His-tagged proteins. Please see the Clontech Related Products section on page (31) for ordering information.


B.subtilisSecretoryProteinExpressionSystem

Features• IncludespBE-SDNA,anE. coli/B. subtilisshuttlevectorwithB. subtilis-derived

subtilisin(aprE)promoter,secretorysignalpeptide(aprESP),MultipleCloningSite,and3’(C-terminal)His-tagsequence

• SuppliedwithSPDNAMixture,alibraryofDNAsequencesencoding173uniquesecretorysignalpeptidesthatcanbeinsertedupstreamofyourtargetgene

• FullycompatiblewithIn-Fusioncloningkitsandsystemstoallowrapidandeasyconstructgeneration

• IncludesB. subtilisstrainRIK1285

Applications• Expressionofsoluble,recombinantproteinsecreteddirectlyintotheculturemedia• Proteinexpressioninahostamenabletomedium-andlarge-scalefermentationin

additiontosmall-scaleculturing• Expressionofproteinswithcomplexstructure,suchasproteinswithdisulfide(S-S)

bonds• GenerationoftargetproteininahostthatisconsideredtobeGenerallyRegardedAs

Safe(GRAS)bytheU.S.FoodandDrugAdministration• Usefulforproducingeasilypurifiedrecombinantprotein–withproperin-frame

cloning,aC-terminalHis-tagcanaidpurificationfromculturemedia

DescriptionBacillus subtilishasbecomeanincreasinglypopularhostforrecombinantproteinexpression.Withitsabilitytosecreteproteindirectlyintoculturemedia,amenabilitytomedium-andlarge-scalefermentation,lackofcodonbias,anddesignationbytheU.S.FoodandDrugAdministrationasanorganismthatisGenerallyRegardedAsSafe(GRAS),it’snowonderthatthemajorityofindustrially-producedenzymesareexpressedinBacillusspeciessuchasB. subtilis.Optimizationofsecretion,however,canbenecessarytoachievehighestyields.Toaddressthis,theB. subtilisSecretoryProteinExpressionSystemfromTakaraBioallowsrapiddevelopmentofalibraryofB. subtilisclones,eachbearingapBE-SconstructinwhichtheORFforyourproteinofinterestisfusedwithsequencesfor173uniquesignalpeptides.Performadownstreamassaytoidentifyandselectcloneswhichsecretethehighestamountoffunctionalproteinintotheculturemedia,andyoucanquicklyidentifythesignalpeptidethatresultsinefficientexpressionofyourdesiredsecretedprotein.

Kit Components*1

SPDNAmixture(0.032pmol/μL)*2 45μLpBE-SDNA(0.5μg/μL)*3 20μgB. subtilisRIK1285*4(glycerolstock) 100μLx2

*1: Librarydevelopment(10reactions)*2: DNAmixtureencodingsecretorysignalpeptidesfromthe173typesofB. subtilis

forusewithIn-Fusioncloningsystem(10reactions).Forthesequenceofsecre-torysignalpeptides,pleaseseetheproductpagefortheB. subtilisSecretoryProteinExpressionSystemontheTakaraBiowebsite.

*3: inTEbuffer(pH8.0)*4: Marburg168derivative:trpC2,lys1,aprEΔ3,nprR2,nprE18

TheSystemprovidessufficientreagentsfor10librarydevelopmentreactions.

Storage• SPDNAmixtureandpBE-SDNA:–20°C• B. subtilis RIK1285glycerolstock:–80°C

Related In-Fusion Cloning ProductsForrapidandeasycloning,usetheIn-FusionHDCloningSystem*(ClontechCat.#639645/639646/639692/639647)orIn-FusionHDCloningSystemCE*(ClontechCat.#639636/639637/639693/639638)togeneratepBE-Sconstructswithyourinsertofinterest.

*:AvailableintheU.S.only.OutsideoftheU.S.,usetheIn-FusionHDCloningKit(ClontechCat.#639648/639649/639650)orIn-FusionHDCloningKitw/CloningEnhancer(ClontechCat.#639633/639634/639635)incombinationwithhigh-efficiencyStellarCompetentCells(ClontechCat.#636763/636766),aHST08E. colistrain.AvailabilityofIn-Fusionsystemsandkitsvariesbygeographiclocation;checkforproductssoldinyourregion.

B. subtilis Secretory Protein Expression System 3380 10 rxns

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Figure 1. Flowchart of the experimental procedure for the B. subtilis Secretory Protein Expression System.

aprEpromoter

Ampr

pBE-S DNA(5,938 bp)

aprE SP

multi cloning site (MCS)

ColE1 ori

pUB ori

Kanr

Mlu I

Eco52 I

His-Tag

173 different types of SP DNA are inserted into this region in place of the aprE SP

Figure 2. Vector map for pBE-S DNA, a B. subtilis/E. coli shuttle vector used with the B. subtilis Secretory Protein Expression System.

Figure 3. Results of measuring β-glycosidase activity of 470 clones of an expression library with different signal peptide sequences.Clonesshowingactivitylevelsofvaryingstrengthswereobserved.ThearrowheadsindicatetheexpressionlevelobservedwiththeaprEsignalpeptide.

A405

321 331 341 351 361 371 381 391 401 411 421 431 441 451 46100.511.522.53

A405

161 171 181 191 201 211 221 231 241 251 261 271 281 291 301 31100.511.522.53

A405

1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 15100.511.522.53

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pColdExpressionVectors

Features• Greatfordifficultproteinsthatcan’tbeexpressedwiththeT7system• Facilitatesincreasedsolubilityduetoexpressionatreducedtemperature• Facilitatesincreasedpurityduetorepressedexpressionofhostproteins

Application• High-efficiencyproteinexpressionusingacoldshockpromoter

DescriptionTakara’spColdExpressionVectorsofferColdShockexpressiontechnologyforhighpurity,highyieldproteinproduction.

ThepColdseriesincludesfourdifferentvectors.EachincludestheColdShockProteinA(cspA)promoterforexpressionofhighlypurerecombinantproteinin E. coli athighyield. Thesevectorsselectivelyinducetargetproteinsynthesisatlowtemperature

(15°C),aconditionatwhichhostproteinsynthesisissuppressedandproteaseactivityisdecreased.Thisresultsinhighyieldsoftargetprotein(~60%ofintracellularprotein).InadditiontothecspApromoter,allfourvectorscontainalac operator(forcontrolofexpression),ampicillinresistancegene(amp r ),ColE1originofreplication,M13IGfragment,andmultiplecloningsite(MCS).Threevectorsalsocontaineitheratranslationenhancingelement(TEE),His-Tagsequence,and/orFactorXacleavagesite.Thesevectorsworkequallywellforsynthesisofnon-labeledandradiolabeledproteinsandcanbeusedwithTakara’sChaperonePlasmidSet(Cat.#3340).

Related ProductsChaperonePlasmidSet,3340,p.15.

Reference1. Quing,G.,et. al.(2004)Nature Biotechnol. 22(7):877-882.

M13 IG

pCold I4.4kb

pCold II4.4kb

pCold III4.4kb

pCold IV4.4kb

cspA 3'UTRmultiple cloning siteFactor Xa siteHis•TagTEEcspA 5'UTRlac operatorcspA promoter

ColE1 ori

Am

p la

cI

M13 IG

cspA 3'UTRmultiple cloning siteHis•TagTEEcspA 5'UTRlac operatorcspA promoter

ColE1 ori

Am

p

Am

p

Am

p

lacI

M13 IG

cspA 3'UTRmultiple cloning siteTEEcspA 5'UTRlac operatorcspA promoter

ColE1 ori

lacI

M13 IG

cspA 3'UTRmultiple cloning sitecspA 5'UTRlac operatorcspA promoter

ColE1 ori

lacI

Inthefollowingexamples,genesthatwerepoorlyexpressedorthatproducedinsolubleproteinwiththeT7promoterexpressionsystemwereexpressedusingthepColdsystem.pColdIDNAwasusedasanexpressionvectorinE. coli.ExpressionfromT7promoter-drivenvectorswasinducedwithIPTGandT7plasmid-containingcellswereculturedat37°C.

Figure 1. Expression of human gene A. HumangeneA(~31kDa)wasexpressedinboththeT7systemandthecold-shockexpressionsystem.NoexpressionwasobservedintheT7system,buthumangeneAwasexpressedinthepColdsystem.

Figure 2. Expression of human gene C. ComparisonofexpressionofsolublehumangeneCprotein(~80kDa)inthecold-shockexpressionsystemvs.theT7systemwasperformed.TargetproteininthesolublefractionofpColdcellswasdramaticallyhigherthanthatoftheT7system.

N.C T7 pColdkDa

97.4

66.2

45

31

21.5

14.4

← Expression enabled

CBB staining of the entire protein fraction

T7 pCold T S T S

kDa

97.4

66.2

45

31

21.5

14.4

T: Entire protein fractionS: Soluble fraction

← Expression level increased

CBB staining

pCold Vector Set 3360 1 Set (ea. 5 µg) pCold I DNA 3361 25 µg pCold II DNA 3362 25 µg pCold III DNA 3363 25 µg pCold IV DNA 3364 25 µg

Takara’s pCold expression vectors

3

Pro

du

ctsforIn

crease

dP

rote

inY

ield

an

dP

urity


Application• Highlyefficientproteinexpressionusingcoldshocktechnology• HighyieldofactiveproteinduetoTriggerFactorchaperoneasasolubility-promoting

fusiontag

DescriptionTakara’spColdTFDNAVectorisafusioncoldshockexpressionvectorthatexpressesTriggerFactor(TF)chaperoneasasolublefusiontag.TriggerFactorisa45kDaprokaryoticribosome-associatedchaperoneproteinthatfacilitatesco-translationalfoldingofnascentpolypeptides.BecauseofitsE. coliorigin,TFishighlyexpressedinE. coliexpressionsystems.ThepColdTFDNAVectorconsistsofthecspApromoterplusadditionaldownstreamsequencesincludinga5’untranslatedregion(5’UTR),atranslationenhancingelement(TEE),aHis-Tagsequence,andamultiplecloningsite(MCS).AlacoperatorisinserteddownstreamofthecspApromotertoensurestrictregulationofexpression.Additionally,recognitionsitesforHRV3CProtease,Thrombin,andFactorXaarebetweenTF-TaqandtheMultipleCloningSite(MCS).Thesesequencesfacilitatetagremovalfromtheexpressedfusionprotein.MostE. colistrainscanserveasexpressionhosts.

pColdTFVector

Figure 1. Expression of protein A in T7 and pCold systemsTheexpressionofenzymeproteinA(~29kDa)waspoorwhenutilizingaT7orpColdIexpressionsystems,evenwhenthepColdIconstructwasco-expressedwithachaperone.Incontrast,theexpressionoftargetproteinasafusion(29kDa+52kDa)wassuccessfulwithpColdTFDNA,andmostoftheexpressedproteinwasinsolubleform.TheexpressedenzymeproteinAshowedactivityevenintheformofafusionprotein(datanotshown).

pCold TF pColdpCold +

Chaperone T71 2 1 2 1 2 1 2

kDa

97

66

45

31

22

1. Cell extract solution

2. Soluble fraction

target protein

co-expressed

trigger factor**

pCold TF DNA 3365 25 µg

Features• Facilitateshighyieldproteinexpressionwithoptimizedproteinfolding• Enablesexpressionoffusionproteinswithasolubletagtooptimizesolubility

DescriptionThepColdProS2expressionvectorfeaturesProteinS,asolubletagfromMyxococcusxanthusfusedtotheN-terminusoftargetproteins.Tightregulationofproteinexpressionismaintainedbyalacoperatordownstreamofthecoldshockpromoter.HRV3CProtease,Thrombin,andFactorXarecognitionsitesareencodedbetweentheProteinStagandtheMSCtofacilitatetagremoval.

pColdProS2DNA pCold™ ProS2 DNA 3371 25 µg

3

Pro

du

cts

for

Incr

ease

dP

rote

inY

ield

an

dP

uri

ty

Application• Site-dependentcleavageofssRNA

DescriptionMazFisatoxinproteininthetoxin-antitoxinmoduleofE.coli.Itpossessesendoribonucleaseactivityandspecificallycleavessingle-strandedRNAatthe5’endof5’-ACA-3’sequences.Thisenzymedoesnotcleavedouble-strandedRNA,double-strandedDNA,orsingle-strandedDNA.mRNAInterferase-MazFissuppliedasafusionproteinofE. coli MazFandTriggerFactor.TheTriggerFactorproteinisanE.colichaperoneprotein.Theenzymeisalsosuppliedwitha5XMazFbuffer(200mMSodiumphosphate,pH7.5,0.05%Tween20.)

mRNAInterferase™-MazFEnzyme mRNA Interferase™-MazF 2415A 1000 units


SPPSystem

Application• Preferentialexpressionoftargetproteinbysuppressionofendogenousproteins

usingmRNAinterferaseplasmid

DescriptionThissystemutilizesanE.coliprotein,MazF,describedasanmRNAinterferasebySuzukiet.al.MazFisasequence-specificendoribonucleasethatcleavessinglestrandRNAsat5’-ACA-3’(ACA)sequences.WhenusingtheSPPSystem,thetranscriptofinterestshouldthereforelackACAsequences.MazFisco-expressedintheE.colihostandsuppressesexpressionofnon-targetgenesbycleavinghosttranscriptsatACAsequences.Therefore,thetargetproteinisthemostabundantlyexpressedprotein(Figure1).BecauseoftherequirementfortargetgenetranscriptstolackACAsequences,theSPPSystemisnotsuitableforallgenesofinterest;however,whenappropriate,itcanresultinextremelyhighlevelsofproteinproduction.

ReferenceSuzuki,M.,et. al.(2005)Molecular Cell18(2)253-261.

Figure 1. Synthesis of cspA-promoter expressed envAB in presence and absence of MazF. E. coliBL21cellsco-expressingMazFandpCold(SP-4)envZBshowedgoodenvZBexpressionandextremelylowbackgroundsynthesisofhostproteins.

SPP System™ I 3367 1 kit SPP System™ II 3368 1 kit SPP System™ III 3369 1 kit SPP System™ IV 3370 1 kit SPP System™ I-IV 3366 1 kit 3

Pro

du

ctsforIn

crease

dP

rote

inY

ield

an

dP

urity


NeoR

SV40 promoter

AmpR

SV40 polyA EF1α promoter

HSV TK polyA

Cla I

EcoR V

EcoR I

BamH I Xba ISse8387 I

Hind III

Cla IEcoR V

pBApo-EF1α Neo5,167 bp

PurR

SV40 promoter

AmpR

SV40 polyA EF1α promoter

HSV TK polyA

Cla I

EcoR V

EcoR I


Hind III

Cla IEcoR V

pBApo-EF1α Pur4,972 bp

Features• Generalpurposegeneexpressionvectorformammaliancells• CanbeusedtoexpressmiRNAprecursorsandothertranscriptionproducts• Allowseasytransferofexpressioncassettetoadenovirusvector

DescriptionpBApo-CMVisageneralpurposegeneexpressionvectorformammaliancells.Thisvectorhasapromoterfromcytomegalovirus(CMVIEpromoter),apoly(A)signalfromthymidinekinaseofherpessimplexvirus(HSV),andamultiplecloningsite(MCS).ThisvectorcanbeusedtoexpressmiRNAprecursorsandothertranscriptionproductsinadditiontoprotein-codinggenes.Thecassettepromoter+ORF+poly(A)signalcassettecanbeeasilytransferredfromthisvectortoanadenovirusvector.

Withtheabilitytoachieveahighinfectionefficiencyacrossabroadspectrumofcelllines,adenovirusvectorsaresuitableforin vitroandin vivogenetransduction.Forconstructingrecombinantadenoviruses,useAdenovirusExpressionVectorKit(DualVersion)Ver.2(Cat.#6170).

Inadditiontoabasicvector(Cat.#3242),thepBApo-CMVseriesalsoincludesvectorswithaneomycinresistancecassette(Cat.#3240)orapuromycinresistancecassette(Cat.#3241)forstableexpressioninmammaliancells.

pBApo-EF1alpha

Features• EF1alphapromoterdirectshigherlevelofexpressionthanCMVIEpromoter• CanbeusedtoexpressmiRNAprecursorsandothertranscriptionproducts• Allowseasytransferofexpressioncassettetoadenovirusvector

Application• Geneexpressioninmammaliancells

DescriptionThepBApo-EF1alphaseriesincludessimplegeneexpressionvectorsformammaliancells.Thesevectorsincludeapromoterfromhumanpolypeptidechainelongationfactor(EF-1alphapromoter)andapoly(A)signalsitefromherpessimplexvirusthymidinekinasegene.TheycanalsobeusedtoexpressmiRNAprecursorsandothertranscriptsinadditiontoprotein-encodinggenes.Versionswithneomycinorpuromycinresistancecassettesareavailable.Apromoter+ORF+poly(A)signalcassettecanbeeasilyremovedfromthesevectorsandtransferredtoanadenovirusvector.Adenovirusvectors,whichhavehighinfectionefficiencyandawidetargetcellspectrum,aresuitableforin vitroandin vivogenetransductions.Forconstructingrecombinantadenoviruses,theAdenovirusExpressionVectorKit(DualVersion)Ver.2(Cat.#6170)isrecommended.

pBApo-EF1alpha Neo DNA 3243 20 µg pBApo-EF1alpha Pur DNA 3244 20 µg

NeoR

PurR

SV40 promoter

AmpR

SV40 polyA CMV IE promoter

HSV TK polyA

Cla I

EcoR V

EcoR I


Hind III

Cla IEcoR VpBApo-CMV Neo

pBApo-CMV Puror

CMV IE promoter

AmpR

HSV TK polyA

Cla IEcoR V EcoR I

BamH IXba I

Pst I/Sse8387 I

Hind III

Cla I

EcoR V

pBApo-CMV

Sal IAcc I

Hinc II

Sph I

pBApo-CMVVectors

pBApo-CMV Neo DNA 3240 20 µg pBApo-CMV Pur DNA 3241 20 µg pBApo-CMV DNA 3242 20 µg

4

Mam

malian

Exp

ress

ion

Vect

ors

Figure 1: Expression of DsRed-Express with pBApo-CMV Neo. HEK293cellsweretransfectedwithapBApo-CMVNeoconstructbearingaDsRed-Expresscassette.Thecellswerevisualizedbyfluorescencemicroscopy2daysaftertransfection.

0

10

20

30

40

50

60

70

ES-E14TG2a

% p

ositi

ve

%positive

EFp

CMVp

0

50

100

150

200

250

300

ES-E14TG2a

MFI

MFI

EFp

CMVp

0

10

15

20

25

30

35

40

ES-E14TG2a

% p

ositi

ve

%positive

EFp

CMVp

5

0

10

20

30

40

50

60

ES-E14TG2a

MFI

MFI

EFp

CMVp

Transient expression Stable Expression

Figure 2: Expression levels of pBApo-EF1α Neo or pBApo-CMV Neo. ExpressionofAcGFP1wasassessedinmouseEScellstransientlyorstablyexpressingthetargetgeneunderthedirectionofeithertheEF1αpromoterorCMVIEpromoter.


4

Mam

malia

nE

xpre

ssion

Vecto

rs

pDON-AI-2

Features• Enableshighlyefficientgenetransduction• RetroviralvectorthatlacksallMoMLV-derivedgenes(gag,pol,orenv)exceptLTR

andpackagingsignal(Ψsequence)• Includesahumanactin-derivedintronandspliceacceptorforefficienttargetgene

expression

Application• Retrovirus-mediatedgenetransferintomammaliancells

DescriptionTheretroviralvectorpDON-AI-2andpDON-AI-2-NeodonotcontainanyMoMLVderivedgenes(gag,pol,orenvcodingsequences)exceptLTRandpackagingsignal(psisequence).TheU3regionof5’LTRhasbeensubstitutedwithastrongerpromoterderivedfromcytomegalovirus,givingthesevectorsahightranscriptionefficiency

andallowingthemtobeusedtogeneratehightiter-recombinantretrovirusesandaccordinglyefficientgenetransductions.Moreover,theycarryahumanactin-derivedintronandspliceacceptorupstreamofthecloningsitetoincreasetheefficiencyoftargetgeneexpressionaftergenetransduction.pDON-AI-2NeoDNA(Cat.#3653)hasaneomycinresistancegeneasadrugselectionmarker.

pDON-AI-2 DNA 3654 20 µg pDON-AI-2 Neo DNA 3653 20 µg

pDON-AI-24586 bp

Ampr

5' LTR

HCMV IE promoter

MLV RMLV U5

SD

ψ

Intron + SA

MCS

PmaCI (1261)Apa I (1266)

Not I (1272)Sac II (1270)

Bgl II (1280)Cla I (1286)Bam HI (1292)Sal I (1298)Hpa I (1304)

MLV U3

MLV R

MLV U5 3' LTR

pDON-AI-2 Neo5719 bp

HCMV

MLV R MLV U5

Intron + SA

MCS

Minimal

Neo

MLV U3MLV R

MLV U5

Amp

r

IE promoter

SV40 promoter

r

PmaCI (1261)Apa I (1266)

Not I (1272)Sac II (1270)


5' LTR

3' LTR

ψ

SD

pMEI-5

Features• Forhighlyefficienttranscription• Toincreasetargetgeneexpressionaftergenetransduction,includesahumanEF1α-

derivedintronwithhighsplicingactivityupstreamofthecloningsite


DescriptionTheretroviralvectorspMEI-5andpMEI-5-NeopossessLTRandpsi(viralpackagingsignal),butlackthestructuralgenesnecessaryforparticleformationandreplication(gag,pol,andenv).BecausethesevectorscontainahumanEF1α-derivedintronwithhighsplicingcapacity,theyfacilitatehightranscriptionefficiency.pMEI-5NeoDNA

containstheneomycinresistancegeneasaselectivemarker.Geneexpressionlevels2-to8-foldhigherthanpDON-Al-2seriescanbeexpected.

pMEI-5 DNA 3656 20 µg pMEI-5 Neo DNA 3655 20 µg

pMEI-54689 bp

MLV U3

MLV RMLV U5

Intron + SA

MLV U3MLV R

MLV U5

Ampr

MCSPmaCI (1760)Apa I (1765)

Not I (1771)Sac II (1769)


ψSD

Xho I (1811)

5' LTR

3' LTR

pMEI-5 Neo5820 bp

MLV U3MLV R

MLV U5

Intron + SA

MCS

minimal

Neo

MLV U3MLV R

MLV U5

Amp

SV40 promoter

r

r

ψ

SD PmaCI (1760)Apa I (1765)

Not I (1771)Sac II (1769)


5' LTR

3' LTR


Features• Highgenetransductionandhightranscriptionefficiency• Enablestheproductionofhightiterrecombinantretroviruses


DescriptionThepDON-5andpDON-5Neovectorsfacilitatebothhigh-efficiencygenetransductionandhighexpression.Theyallowhigherproductionofhigh-titerrecombinantretrovirusesthanpMEI-5orpMEI-5-Neo(seeFigure1).BytransfectingthevectorpDON-5orpDON-5Neovectorintoanappropriatepackagingcellline,thevectorexpressestransientorstabletranscribedproductcontainingviruspackagingsignal(psi)andtargetgeneandselectivemarker.

ThesevectorspossessLTRandpsi(viralpackagingsignal),butnotthestructuralgenesnecessaryforparticleformationandreplication(gag,pol,andenv).Thesevectorsincludeastrongcytomegaloviruspromoter(HCMVIE)withintheU3regionof5’LTRandahumanEF1α-derivedintronwithhighsplicingactivityupstreamofthemultiplecloningsite.pDON-5Neoincludestheneomycinresistantcassetteasaselectivemarker.

pDON-5

pDON-5 DNA 3658 20 µg pDON-5 Neo DNA 3657 20 µg

pDON-54697 bp

Ampr

5' LTR

HCMV IE promoter

MLV RMLV U5

SD

ψ

Intron + SA

MCS

MLV U3

MLV R

MLV U5 3' LTR

PmaCI (1370)Apa I (1375)Sac II (1379)Not I (1381)Bgl II (1389)Cla I (1395)Bam HI (1401)Sal I (1407)Hpa I (1413)Xho I (1421)

pDON-5 Neo5828 bp

HCMV

MLV R MLV U5

Intron + SA

MCS

Minimal

Neo

MLV U3MLV R

MLV U5

Amp

r

IE promoter

SV40 promoter

r

PmaCI (1370)Apa I (1375)

Not I (1381)Sac II (1379)


5' LTR

3' LTR

ψ

SD

4

Mam

malian

Exp

ress

ion

Vect

ors

0

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

6,000,000

7,000,000

8,000,000

ivp/

ml

DON-AI-2DON-AI-2Neo

MEI-5 Neo MEI-5 DON-5 Neo DON-5

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

DON-AI-2DON-AI-2Neo

MEI-5 Neo MEI-5 DON-5 Neo DON-5

Figure 1: Comparison of Virus Titers (ZsGreen/HT1080)

TheZsGreengenewasinsertedintotheBamHI/HpaIsiteoftheindicatedvectors.G3T-hicellsweretransientlytransfectedandthen2setsofrecombinantretroviruseswereproducedforeachviralvector.

Figure 2: ZsGreen Expression Intensity(relativevaluetothemeanfluorescenceintensityof1copy/cell)HT1080cellswereinfectedwithrecombinantretrovirusesatvariousdilutionratesusingpolybrene.Threedaysaftertransduction,genetransferefficiencyandZsGreenexpressionintensityweremeasuredusingaflowcytometer.ValuesshownwerenormalizedusingtheexpressionintensityofDON-AI-2virusas1.


HumangeneA(~70kDa)wasexpressedininsolubleformwhenusingpColdIalone.However,thelevelofsolubleexpressedproteinincreasedsignificantlywhenthechaperoneplasmidpG-Tf2wasco-expressedwiththepColdIconstruct.

HumangeneB(~24kDa)wasnotexpressedwhenusingpColdIDNAalone.However,co-expressionwiththechaperoneplasmidpG-Tf2resultedinexpressionofhighlevelsoftargetproteininsolubleform.

ChaperonePlasmidSet

Application• Promotescorrectin vivo foldingofexpressedrecombinantproteinsinE. coli

DescriptionTheChaperonePlasmidSetconsistsof5differentplasmids,eachofwhichisdesignedtoexpressmultiplemolecularchaperones.Together,theyfunctionasa“chaperoneteam”tofacilitateproteinfolding.Co-expressionofatargetproteinwithoneoftheseplasmidsincreasestherecoveryofsolubleproteins.EachplasmidcarriesanoriginofreplicationderivedfrompACYCandaCmrgene,whichallowsusewithE.coliexpressionsystemsutilizingColE1-typeplasmidswithanampicillinresistancegeneasamarker.ThechaperonegenesaresituateddownstreamofanaraBorPzt-1(tet)promoter.Therefore,expressionoftargetproteinsandchaperonescanbeinducedindividuallyifthetargetgeneisplacedunderthecontrolofotherpromoters(e.g.lac).Theseplasmidsalsocontainthenecessaryregulator(araCortetR)foreachpromoter.

Notethatthissystemcannotbeusedincombinationwithchloramphenicol-resistantE.colihoststrainsorexpressionplasmidsthatcarryachloramphenicol-resistancegene.Forexample,E.coliBL21(DE3),whichisoftenusedwithpETsystems,isacompatiblehoststrain.However,E.coliBL21(DE3)pLysSandBL21(DE3)pLysE,whichcontainpLysSorpLysEplasmidsthathavethepACYCreplicationoriginandtheCmrgene,cannotbeusedwiththissystem.

PlasmidswiththeChaperonePlasmidSetworkwellincombinationwiththepColdexpressionsystemvectors.

Kit Components5plasmids:conc.10ng/µL;100µLeachplasmid

Resistance

Plasmid Chaperone Promoter Inducer Marker pG-KJE8 dnaK-dnaJ-grpE-groES-groEL araB,Pzt1 L-Arabinose,TetracyclineCmr

pGro7 groES-groEL araB L-Arabinose Cmr

pKJE7 dnaK-dnaJ-grpE araB L-Arabinose Cmr

pG-Tf2 groES-groEL-tig Pzt1 Tetracycline Cmr

pTf16 tig araB L-Arabinose Cmr

References1. Nishihara,K.,etal.(2000)Microbiol.66(3):884-889.

2. Nishihara,K.,etal.(1998)Appl.Environ.Microbiol.64(5):1694-1699.

pKJE77.2 kb

Cmr

araC pACYC ori

araB

dnaK dnaJ

grpE

pACYC ori

groEL

pGro75.4 kb

Cmr

araC

araB

groES

rrnBT1T2

pACYC ori

Pzt1pG-KJE8

11.1 kb

CmraraC

tetR

araB

dnaK

dnaJgrpE

groES

groEL

pACYC ori

tig

pTf165 kb

Cmr

araC

araBpACYC ori

pG-Tf28.3 kb

Cmr

tetRtig

groES

groEL

Pzt1

ThecombinationofColdShockExpressionVectorsandtheChaperonePlasmidSetoftenleadstosignificantimprovementinexpressionlevelofsolubleformsoftargetproteins.IfsufficientexpressionorsolubilizationcannotbeachievedusingpColdvectorsalone,werecommendco-expressionwithchaperoneplasmids.Furthermore,pColdvector-basedexpressionsystemsmayproducebetterresultsbyco-expressingchaperoneplasmidscarryingthetigsequence,suchaspG-Tf2orpTf16,whichareincludedintheChaperonePlasmidSet(datanotshown).

Chaperone Plasmid Set 3340 1 Set

5

Fold

ing


Guanidine hydrochloride

unfolds inclusion bodies

Surfactants prevent protein

aggregation

Highly polymerized CA removes surfactants and facilitates protein

refolding

Biologically active protein in

thermodynamically stable native conformation

RefoldingCAKit

Application• Refoldingofisolatedinclusionbodyproteins

DescriptionTheRefoldingCAKitusesanovelartificialchaperonetechnology(licensedfromNFRI,BTRAI,andEzakiGlicoCo,Ltd.)inaneasy2-stepprocedureforoptimizingtherefoldingconditionsofinclusionbodyproteins.Optimizationallowsidentificationofthebestconditionsforcorrectproteinfoldingandrestorationofproteinactivity.

TheSmallKit(Cat.#7350)issuppliedwithguanidinehydrochlorideandDTTforproteindenaturation,fourdifferentsurfactantsthatcanbeaddedindependentlytotheunfoldedproteinsolutiontoprotectagainstmolecularaggregation,andhighlypolymerizedcycloamylose(CA),anartificialchaperone,forsurfactantremovalandrecoveryofproteinactivity.OvernightincubationoftheCA-treatedproteinisfollowedbyaquick10-minutecentrifugation.Theresultingsupernatantcontainstherefoldedprotein.

TheLargeKit(Cat.#7351)isusedforlarge-scalerefoldingafterinitialdeterminationofwiththeSmallRefoldingCAkit,andconsistsonlyofdenaturantandCA.

Related ProductsChaperonePlasmidSet,3340,p.15.

Kit Components

7350 (Small Kit)8Mguanidinehydrochloride(GdmCl) 2x1mL4Mdithiothreitol(DTT) 50µL4surfactants:

1%Tween40 2x1mL1%Tween60 2x1mL1%CTAB(cetyltrimethylammoniumbromide) 2x1mL1%SB3-14(myristylsulfobetaine) 2x1mL

200mMDL-cystine 2x0.75mL3%CA(highlypolymerizedcycloamylose) 7x1.6mL

7351 (Large Kit)3%CA(highlypolymerizedcycloamylose) 6x20mL8Mguanidinehydrochloride(GdmCl) 2x10mL

References1. Machida,S.,etal.(2000)FEBSLett.486(2):131-135.

2. Sundari,C.S.,etal.(1999)FEBSLett.443(2):215-219.

3. Daugherty,D.L.,etal.(1988)J.Biol.Chem273(51):33961-33971.

Principle of the Refolding CA Kit

Refolding CA Kit 7350 (small) 25 reactions Refolding CA Kit 7351 (large) 1 kit

5

Fold

ing

ChaperoninGroE

Application• Facilitatesrefoldingofdenaturedproteins

DescriptionChaperoninGroEisaproteincomplexcomposedofGroEL(14subunits,57kDa)andGroES(7subunits,10kDa).Itisthoughttosupporttheabilityofproteinstoformtertiarystructureuponorimmediatelyaftertranslation.GroEisessentialtoassembly(andpresumablyreassemblyafterdenaturation)ofproteincomplexesin vivo.ChaperoninGroELandGroEScanbeusedforrefoldingdenaturedproteinstorecoverfunctionalactivity.

Application• Facilitatesproteinrefoldingbypromotingexchangereactionsbetweendisulfide

bonds

DescriptionCorystein™(Purothionin)Reagentisapolypeptidepurifiedfromwheatendosperm.Itcatalyzestheformationofcorrectdisulfidebondsinproteins.Corystein™Reagentcanbeusedaloneortogetherwiththioredoxinonavarietyofproteinstore-formdisulfidebonds.

Corystein™(Purothionin)Reagent

Chaperonin Gro EL 7330 5 mg Chaperonin Gro ES 7331 0.5 mg

Corystein™ (Purothionin) Reagent 7311 5 mg


High-levelSecretionofRecombinantProteinusingtheBrevibacillusExpressionSystemBy Michikazu Tanio and Toshiyuki Kohno, Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan

IntroductionAvarietyofcurrentlyusedheterologousproteinexpressionsystemshasbeendeveloped,suchasE. coli,yeast,insectcells,animalcellsandcell-freesystems.Suchexpressionsystemsenabletheproductionofproteinsthataredifficulttoisolatefromrawmaterial,andallowintroductionofmutations,heavyatoms,andisotopes.ThemostpopularandfirstchoiceinmostcasesistheE. coliproteinexpressionsystem,whichfeatureshighproductivity,easeofuse,andrelativelylowcost.However,manyproteinsareoftenproducedinE.colisystemsininsolubleinclusionbodies,particularlyforproteinswithdisulfide(S-S)bonds,secretedproteins,orproteinsoriginatingfromhigherorganisms.Refoldingofinclusionbodiesoroftargetproteinssecretedtotheperiplasmcanbeperformed,butthisgenerallyleadstolowerproductionyieldsofactiveprotein.

TheyeastproteinexpressionsystemusingPichia pastorisisknowntobequiteefficientforexpressionofproteinscontainingS-Sbonds,butexpressionlevelsarehighlyvariableamongtargetproteins.

Varioustypesofproteinshavebeensuccessfullyexpressedusinginsectcellsoranimalcells,includingtargetproteinsthatcouldnotbeexpressedwithE. colisystems,butE. coliremainssuperiorforproductivityandcost.However,amajoradvantageofeukaryoticexpressionsystemsincludingyeastisthecapacityforpost-translationalmodificationsuchasphosphorylationorglycosylation,althoughsuchmodificationswouldbeanobstacleinstructuralanalysisbyleadingtoproteinheterogeneity.

Cell-freeexpressionsystemscansometimesexpresscytotoxicproteins,andavoidmetabolicissueswhenperformingselectiveamino-acidlabelingwithstableisotopes.However,cell-freereactionsareperformedinareductiveenvironment,thuspreventingexpressionofproteinscontainingdisulfidebonds.Thesesystemsarealsoofrelativelyhighcost.

Thus,eachheterologousproteinexpressionsystemhasadvantagesanddrawbacksandselectionofanappropriatesystemmustconsiderbothcostandresearchpurpose.However,thereisnosatisfactorysystemsuitableforproteinscontainingdisulfidebonds.

Inourlaboratory,wehaveanalyzedproteinstructureandfunctionusingdifferentexpressionsystems.TheBrevibacillusExpressionSystemwasintroducedrecently.Thisexpressionsystem,whichTakaraBiolaunchedin2006,hasalreadyshownnumeroussuccessesforproducingsecretedrecombinantproteins(1-4).WiththissystemwecouldobtainsomesecretedorcytoplasmicproteinsthatfailedtobeexpressedwithanE. coli systemorayeastsystem(5-7).ConsideringthesimpleprotocolandaffordablecostoftheBrevibacillusExpressionSystem,itshouldbeafirstchoice-beforetheE. coliexpressionsystem-forrecombinantproteinexpression.

Inthispaper,wedescribebothrecombinantproteinproductionandstableisotopelabelingusingtheBrevibacillusExpressionSystem.

MethodsExpression and Purification of Two Recombinant Proteins - Human M-Ficolin Recognition Domain (FD1: Molecular weight 26.8 kDa) and Human FK506 Binding Protein (FKBP: Molecular weight 13 kDa)

Construct DesignAfteramplificationbyPCRusingHumanUniversalQUICK-Clone™cDNAII(Clontech)asatemplate,eachcDNAencodingthetargetproteinwasclonedintothepNCMO2vectorwithinsertionofaC-terminal6xHis-Tag.TheexpressionvectorconstructsweretransformedintoBrevibacillus choshinensisElectro-Cellswiththeelectroporationprotocolrecommendedbythemanufacturer'susermanual.

Analysis of ExpressionTheobtainedcolonieswereculturedfor1-3dayswith3-5mlofmediaina14-18mmdiametertube(37°C,200rpmreciprocalshakingculture).Twokindsofmediawereprepared:2SYNm(2%glucoseand4%soytoneand0.5%yeastextractand1mMcalciumchlorideand50μg/mlneomycin)andTMNm(1%glucoseand1%polypeptoneand0.5%bonitoextractand0.2%yeastextractand0.001%ironsulfateand0.001%manganesesulfate,0.0001%zincsulfate,and50μg/mlneomycin).ExpressionanalysisbySDS-PAGEandwesternblotwithanti-HisantibodyshowedthatFD1couldbedetectedonlyin2SYNmmediumandFKBPwasproducedinboth2SYNmandTMNmmedia.2SYNmmediumwasselectedforlarge-scalecultureduetoitseaseofpreparation.

Large-Scale Culture and Purification1mlofbacterialpre-cultureincubatedin3-5mlof2SYNmmediumwasaddedtoa500mlflaskwith100mlof2SYNmorSYNmmedium(2%glucoseand0.8%soytoneand0.5%yeastextractand50µg/mlneomycin)andincubatedat27°Cor37°Cat100rpmfor1-5days.Thenumberofcultureflaskswasincreasedasneededaccordingtothedesiredamountofproteinexpressionandtheenduseofrecombinanttargetprotein.TheexpressedproteinswerepurifiedbyaffinitychromatographywithTALON®MetalAffinityResin(Clontech)afteradjustingtheculturesupernatantpHto8.

FD1wassubjectedtoionexchangeandgelfiltrationchromatography(Fig1),andFKBPwaspurifiedbygelfiltrationchromatographyonly.

Stable Isotopic Labeling1mlofthebacterialpre-culturein2SYNmwasaddedinto500mlflaskwith100mlofStableIsotopeLabelingC.H.L.Medium(ChlorellaIndustryCo.,Ltd.)addedby50μg/mlneomycinandincubatedat27-37°C,100rpmfor1-5days.Thelabeledproteinswerepurifiedbyaffinitychromatographyandgelfiltrationchromatography.Foraminoacid-selectivelabeling,theunlabeledC.H.L.mediumaddedbylabeledaminoacids(100mg/L)wasusedasculturemedia.

Figure 1. Expression and purificaton of FD1 protein with the Brevibacillus Expression Sytem. Lane 1:Culturesupernatantafter2days.Lane 2:FD1proteinpurifiedbyTalon.Lane 3:FD1proteinpurifiedbyionicexchangeandgelfiltrationchromatography.

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Results

Secretion by the Brevibacillus Expression SystemFD1,whichcontainstwoS-SbondsandincludesasubstratebindingdomainofhumanM-Ficoline,functionsasaforeignsubstancerecognitionproteinduringinnateimmunity(Fig2).AlthoughwehavealreadyreportedtheFD1crystallographicstructurebysecretionofFD1withtheyeastexpressionsystemP.pastoris(8-10),atthetimeoftheoriginalstudieswecouldhardlyproduceanyFD1derivativesinthisyeastexpressionsystem.WiththeBrevibacillusExpressionSystem,however,wesucceededintheproductionofsecretedFD1proteinandseveralFD1derivativeswithnormallevelsofactivity(6).Withregardtoexpressionconditions,wefoundmostefficientproductionoccurringat200rpmforashakingcultureintesttubesandat100rpmforrotaryshakingculturesinflasks.Weobservedlowerproductionathigherrpmlevels.Themostappropriateculturetemperaturewas37°C.However,inonecaseofacertainFD1variant,theproteinexpressedat37°Cshowednosubstratebindingactivity,eventhoughnormal

bindingactivityoccurreduponcultureat27°C.Thisimpliesthat,especiallyforthederivatives,irreversibledegenerationmightoccurinthestableprotein,eventhoughthetargetproteinhasbeensecretedefficiently.Severalexperimentswithdifferentmediacompositionsshowedthatmediacontaining~0.8%soytonewithoutcalciumchlorideisthemostsuitableforproductionofsecretedproteins.AsimilartrendoccurrednotonlyforFD1butalsoFKBP.Ontheotherhand,pre-culturefromasinglecolonywithSYNmmediaunderthisconditionresultedinnotablylowgrowth.Takingallresultsintoconsideration,

wechoseourfinalcultureconditionforpre-cultureas3-5mlof2SYNmmediumat37°Cwithshaking(200rpm)overnight(15-18h),andforlarge-scalecultureas100mlofSYNmmediumat27°Cwithrotaryshaking(100rpm)forafewdays(2-4days).Undertheseconditions,wecouldobtain~10mgofpurifiedFD1proteinfrom1Lculture,slightlyhigherthanweobtainedusingtheyeastexpressionsystem(5-8mg/L).WithactivityanalysisofFD1derivativeproteinsobtainedusingtheoptimizedcultureconditionsdescribedabovefortheBrevibacillusExpressionSystem,wehavesuccessfullyidentifiedtheresiduesinvolvedinFD1substratebinding(6).

Inotherstudies,FKBP,acytoplasmicproteinlackingS-Sbonds,wassecretedwiththeBrevibacillusExpressionSystem(5,7).ThecultureandpurificationconditionswerealmostthesameasdescribedaboveforFD1aboveexceptthatthetemperatureforlarge-scaleculturewas37°Cinsteadof27°CbecauseofthehigherproteinstabilityforFKBP.TheyieldofthepurifiedFKBPproteinwith2SYNmmediumwasabout16mg/L.

Inourlaboratory,wehavesuccessfullyusedtheBrevibacillusExpressionSystemtoproduceothercytoplasmicproteinsinadditiontoFKBP.OneproteinofE. coliorigin,whichappearedasamixtureofmonomeranddimerduetotheS-Sbondconnectingoneproteinmoleculetoanotherintheoxidizedenvironmentofthemedium,couldbeobtainedasahomogenouspopulationofmonomerswiththisexpressionsystembyadding10mMofdithiothreitol(DTT)duringthepurificationsteps.Inanothercasewithaproteinofhumanorigin,expressionoccurredinthecytoplasmofE. coli,buttheE.coli-expressedproteinwasdegradedduringtheextractionprocesspossiblybecauseofendogenousE.coliproteaseactivity.WiththeBrevibacillusExpressionSystem,however,thisproteinwassuccessfullysecretedwithoutdegradation.TheseresultssuggestthattheBrevibacillusExpressionSystemisusefulnotonlyforsecretoryproteinswithS-Sbondsbutalsofortheexpressionofcytoplasmicproteins.

Stable Isotope LabelingMethodsforstableisotopelabelingwiththeBrevibacillusExpressionSystemhadnotbeenestablishedpriortothestudiessummarizedhere(5-7).Stableisotopelabelingofatargetproteinisanindispensabletechniqueforproteinstructuralandfunctionalanalyses,especiallyforNMRstudies.WetriedtoestablishthestableisotopelabelingwithBrevibacillusExpressionSystem,motivatedtodosobythenecessityofNMRanalysisofFD1.WeusedFKBPasamodelproteinbecausetheNMRsignalsforthatproteinwerealreadyassignedandbecauseFKBPcontainsall20typesofaminoacids.Afterperformingproteinexpressiontestswithseveralbacterialminimalmedia,includingM9minimalmedium,StableIsotopeProbingC.H.L.Medium,manufacturedbyChlorellaIndustryCo.,LTD.,Japan,showedthebestresultsintermsofbothgrowthandofexpression.TheproteinyieldofpurifiedFKBPinC.H.L.mediumwasabout2-13mg/Lculture(at37°Cfor1-2days).Consideringthat1)about91%ofFKBPaminoacidswerelabeledwith15Nwhenusing15NLabelingC.H.L.medium,and2)the1H-15NHSQCNMRspectrumwasquitesimilartothedataobtainedwithE. coliexpressionsystems,wecouldconfirmthatthepurifiedproteinsexpressedwiththeBrevibacillusExpressionSystemmaintainedtheirnormalfoldingstructure.Inaddition,wetestedspecialmediainwhicheachindividualkindof15Nlabeledaminoacidwasaddedtothenon-labeledC.H.L.medium,tryingatotalof19kindsofaminoacidsexceptproline.Asaresultof1H-15NHSQCNMRspectrumanalysisonthelabeledproteinobtainedwiththese19kindsofC.H.L.mediumcontainingeach15Nlabeledaminoacid,wefoundthatselectivelabelingispossiblewiththeBrevibacillusExpressionSystemforninekindsofaminoacidresidues(Table1).Ontheotherhand,theacidicandaromaticaminoacidsaremetabolizedtootheraminoacidsfromthefirstdayofculture.Glycine,isoleucine,leucine,serineorthreonineareeachmetabolizedtootheraminoacids,withtheendproductsbeingspecifictoeachresidue.Wedecidedtoharvestcysteine-labeledFKBPafter1dayofculturebecauseFKBPwasobservedtodegradeafter2daysculture.Suchdegradationwasalsoobservedafter3days cultureinthetyrosine-labeledC.H.Lmedium.

Next,inordertoshowtheresultofstableisotopiclabelingofFD1,wetriedtoperformtheexperimentinasimilarwayasdescribedforFKBPlabeling.WefoundthatwiththesimpleC.H.L.mediumonly,theFD1yieldwasnotasgood,butweobtainedbetteryieldwiththespecialC.H.L.medium(C.H.L.aa)where100mg/Lofeightkindsofamino

Figure 2. Crystal structure of FD1 protein (PDB ID 2D39) (9).

AminoAcids Metabolism MetabolismRate(%)** Selection

Ala - <20 ++

Arg - <35 ++

Asn - <30 ++

Asp almostallaa - -

Csy - <18 ++

Gln - <61 ++

Glu almostallaa - -

Gly Cys,Ser,Trp - +

His - <25 ++

Ile Leu,Val - +

Leu Ile,Val - +

Lys - <26 ++

Met - <16 ++

Phe almostallaa - -

Ser Cys,Gly,Trp - +

Thr Cys,Gly,Ser,Trp - +

Trp almostallaa - -

Tyr almostallaa - -

Val - <23 ++

Table 1. Amino acid selectivity of Brevibacillus* (7). Brevibacillus choshinensisHPD31-SP3.**EstimatedbycomparingaverageNMRsignalsobtainedby1H15NHSQCNMRspectrumresultsof15NlabeledFKBPproteins.

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acids(withoutcysteine),respectively,wereutilizedoutofthenineresiduesthatcanbelabeledselectivelywiththeBrevibacillusExpressionSystem.Therefore,forthepurposeofproducingselectively-labeledFD1protein,wedecidedtousetheC.H.L.aamediumwhereatargetaminoacidislabeledwithstableisotope,ortheC.H.L.aamediumwiththelabeledcysteine.ThisimprovementhasmadetheaminoacidselectivelabelingofFD1possible,andwehavesuccessfullyobtainedseveral1H-15NHSQCNMRspectraonFD1(Fig3A-C).TheproteinyieldofpurifiedFD1was2-5.5mg/L(after5daysofculture).Moreover,degradationofcysteine-labeledtargetproteinwasnotobservedinFD1.Inconclusion,wehaveestablishedasimpleandcost-effectiveStableIsotopeLabelingmethodwiththeBrevibacillusExpressionSystem.

ConclusionsTheproductionofsecretedrecombinantproteinusingtheBrevibacillusExpressionSystemhasseveraladvantages:

1)NormalS-Sbondformationcanbeexpected.

2)Targetproteinscanbepurifieddirectlyfromthemediawithoutneedforbacteriallysis.

3)Alargeamountoftargetproteincanbeobtainedbecausetheyaresecretedintothemediaratherthansequesteredinthebacterialcytoplasm.

4)SomeproteinsthatreadilyforminclusionbodieswhenexpressedinE.coliaremorelikely to maintain their normal folding structure during dilution into the culturemedia.

5)Even cytotoxic proteins that exhibit toxicity inside the cells can potentially beproducedefficientlyusingtheBrevibacillusExpressionSystembybeingsecretedintotheextracellularmedium.

6)The Brevibacillus Expression System offers an affordable alternative to eukaryoticsystems. Although eukaryotic expression systems such as insect cells are primarilyused for the secretory production of recombinant protein at present, eukaryoticsystems requires both sophisticated protocols and expensive equipment comparedwiththeE. coliexpressionsystem.Fromthatperspective,theBrevibacillusExpressionSystem could be recommended as an alternative method for producing proteinsthat cannot be easily expressed by E. coli. The Brevibacillus system is compatiblewithcultureequipmentandmaterialsusedforE. coliexpression.Additionally,NMRstructural analysis of secreted proteins produced with the Brevibacillus ExpressionSystemmaybecomemoreattractiveinthefuture.

However,inourexperience,someproteinswereexpressedatlowlevelsandotherscouldnotevenbeclonedintothevector.Moreover,inordertoimprovetheyieldoftheobtainedprotein,increasingTALONresinvolumeandre-applyingtheflow-throughisalsorequired.Webelievethisimpliesthatsome2SYNmandTMNmmediumcomponentsmaypreventabsorptionofHis-tags.Inthefuture,weexpectthatadditionalstudiesonbothadvantagesanddrawbacksofBrevibacillusExpressionSystemwillleadtofurtherdevelopmentsandimprovementsofthisexpressionsystem.

References1. Udaka, S., Yamagata, H. (1993) High-level secretion of heterologous proteins by

Bacillus brevis.Methods Enzymol.217:23-33.

2. Miyauchi,A.,Ozawa,M.,Mizukami,M.,Yashiro,K.,Ebisu,S.,Tojo,T.,Fujii,T.,Takagi,H. (1999) Structural conversion from non-native to native form of recombinanthuman epidermal growth factor by Brevibacillus choshinensis. Biosci. Biotechnol .Biochem.63(11):1965-1969.

3. Yashiro, K., Lowenthal, J. W., O’Neil, T. E., Ebisu, S., Takagi, H., Moore, R. J. (2001)High-level production of recombinant chicken interferongamma by Brevibacillus choshinensis.Protein Expr. Purif.23(1):113-120.

4. Tanaka,R.,Mizukami,M.,Ishibashi,M.,Tokunaga,H.,Tokunaga,M.(2003)Cloningand expression of the ccdA-associated thiol-disulfide Oxidoreductase (catA) genefrom Brevibacillus choshinensis: stimulation of human epidermal growth factorproduction.J. Biotechnol.103(1):1-10.

5. Tanio,M.,Tanaka,T.,Kohno,T.(2008)15NisotopelabelingofaproteinsecretedbyBrevibacillus choshinensisforNMRstudy.Anal. Biochem. 373(1):164-166.

6. Tanio, M., Kohno, T. (2009) Histidine regulated activity of M-ficolin. Biochem. J. 417(2):485-491.

7. Tanio, M., Tanaka, R., Tanaka, T., Kohno, T. (2009) Amino acid-selective isotopelabeling of proteins for nuclear magnetic resonance study: Proteins secreted byBrevibacillus choshinensis.Anal. Biochem.386(2):156-160.

8. Tanio, M., Kondo, S., Sugio, S., Kohno, T. (2006) Overexpression, purification andpreliminarycrystallographicanalysisofhumanM-ficolinfibrinogen-likedomain.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.62(Pt.7):652-655.

9. Tanio,M.,Kondo,S.,Sugio,S.,Kohno,T.(2007)Trivalentrecognitionunitofinnateimmunity system: crystal structure of trimeric human M-ficolin fibrinogen-likedomain. J. Biol. Chem.282(6):3889-3895.

10Tanio,M.,Kondo,S.,Sugio,S.,Kohno,T.(2008)TrimericstructureandconformationalequilibriumofM-ficolinfibrinogen-likedomain.J. Synchrotron Radiat.15(Pt.3):243-245.

Figure 3. 15N stable isotope labeling with Brevibacillus Expression System. 1H-15NHSQCNMRspectrumresultsofA)FD1proteinwith15N-labeledaminoacids,B)FD1proteinwith[α-15N]labeledAla,andC)FD1proteinwith[α-15N]labeledHis.IthasbeenknownthatFD1proteinhas2S-Sbonds(Fig.2).ThisexperimentwasperfomedwithaFD1variant.(F127S/L128S)staysasmonomerinsolutions,becausewild-typeFD1formstrimers.

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Theelucidationofproteinstructureandfunctioncontinuestobeimportantinthepost-genomicera.Anefficientproteinproductionsystemiscriticalforobtaininglargeamountsofcorrectlyfoldedrecombinantproteinforstudy.E. coliexpressionsystemsareusedextensivelyforproductionofrecombinantproteins,andhavetwomajoradvantagesoverotherexpressionsystems:(1)easeofuse,and(2)lowcost.However,somerecombinantproteinsdonotfoldcorrectlyduringexpressioninE. coli,resultingindepositsofinactiveinsolubleproteintermed“inclusionbodies”.

Series of pCold VectorsIncollaborationwithProf.MasayoriInouye(UniversityofMedicineandDentistryofNewJersey),TakaraBiohasdevelopedthepColdDNAVectors,aseriesofnovelproteinexpressionvectors.ThepColdVectorsprovideincreasedin vivoproteinyield,purity,andsolubilityofexpressedrecombinantproteinsusing“coldshock”technology.ThecspA(coldshockproteinA)promoterandrelatedelementshavebeenincorporatedintothesevectorstoup-regulatetargetproteinproductionatloweredincubationtemperatures(37°C-15°C).Thistemperaturedropalsosuppressesexpressionofothercellularproteins,repressesproteaseactivity,andtemporarilyhaltsoverallcellgrowth.Thisprocessallowsexpressionoftargetproteinsathighyield,highpurity(upto60%ofcellularprotein),andincreasedsolubilityascomparedwithconventionalE. coliexpressionsystems.Co-expressionofoneormorechaperoneproteinsduringexpressionofaheterologoustargetproteinhasproveneffectiveforobtaininghigheramountsofsolublerecombinantprotein(seeTakara’sChaperonePlasmidSet(Cat.#3340)).Thisprocedure,though,lackstheconvenienceofasingletransformationstep.

pCold TF VectorsTakara’spColdTFDNAVectorisafusioncoldshockexpressionvectorthatexpressesamolecularchaperone(TriggerFactor(TF))asasolubletag.TriggerFactorisaprokaryoticribosome-associatedchaperoneprotein(48kDa)thatfacilitatesco-translationalfoldingofnewlyexpressedpolypeptides.BecauseofitsE. coliorigin,TFishighlyexpressedinE. coliexpressionsystems.ThepColdTFDNAVectorconsistsofthecspA(coldshock)promoterplusadditionaldownstreamsequencesincludinga5’untranslatedregion(5’UTR),atranslationenhancingelement(TEE),aHis-Tagsequence,andamultiplecloningsite(MCS).AlacoperatorisinserteddownstreamofthecspApromotertoensurestrictregulationofexpression.Additionally,recognitionsitesforHRV3CProtease,Thrombin,andFactorXaarelocatedbetweentheTF-TagandtheMCStofacilitatetagremovalfromtheexpressedfusionprotein.MostE. colistrainscanserveasexpressionhosts.

pColdTFDNAVectorcombineshigh-yieldcoldshockexpressiontechnologywithTriggerFactor(chaperone)expressioninasinglevectortofacilitatecorrectproteinfolding,thusenablingefficientsolubleproteinproductionforotherwiseintractabletargetproteins.Thefollowingexperimentcomparesresultsgeneratedusing1)pColdI,2)pColdIco-expressedwithaChaperoneplasmid,3)pColdTF,or4)T7promoterconstructstoexpressvariousproteins.

Materials and MethodspColdDNAIandpColdTFDNAcloningandexpressionprocedures*wereconductedasdescribedinthisexperimentaloverview:

1) Insert the target gene to the multiple cloning site of the pCold DNA vector forexpression.

2) TransformtheE. colihoststrain(e.g.BL21)withtheexpressionplasmidandselectforamprtransformants.

3) Inoculate the transformants into medium including 50 µg/mL of ampicillin, andincubatewithshakingat37°C.

4) AtOD600=0.4-0.5,refrigeratethecultureat15°C(withoutshaking)for30minutes.

5) Add IPTG to a final concentration of 0.1-1.0 mM, then continue incubation withshakingat15°Cfor24hours.

6) Collect the cells and confirm the expression of the target protein by SDS-PAGE ofsolubleandinsolublefractionsorbyactivityassay.

ExpressionfromT7promoter-drivenvectorswasperformedusingastandardprotocolutilizingIPTGinductionandsubsequentculturingat37°C.

*Cultivation/inductionconditions(culturemedium,aeration,timingofinduction,concentrationofinducer,cultivationtimeafterinduction)shouldbeoptimizedforeachtargetprotein.

ThepColdTFProteinExpressionSystemProducesSoluble,ActiveProteininE.coli

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Figure 2: Increased Expression of Soluble Protein B Using the pCold Expression System

Trx GST Nus

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Results and DiscussionProteinexpressionusingthepColdTFExpressionVectorwascomparedwithproteinexpressionusing(1)thepColdDNAIVectoralone,(2)co-expressionusingthepColdDNAIVectorwithTakaraBio’sChaperonePlasmidpTf16,and(3)aT7promoterexpressionsystemwhichincludedothersolubilization-promotingtags.

Figure1showsthesuccessfulproductionofenzymeproteinAusingthepColdTFsystem.Uponexpression,thisprotein(estimatedmolecularweight29kDa)wasnotobservedasadiscernablebandwitheithertheT7expressionsystemorevenwithpColdI(byeitherindividualexpressionorchaperoneco-expression).

However,theexpressionofthetargetproteinandtargetplustag(29kDaand52kDa)wasobservedusingpColdTF,andmostoftheobtainedproteinwasinsolubleform.SubsequentassaysconfirmedthattheexpressedenzymeAretainsactivityevenasafusionprotein(datanotshown).

Figure2showsimprovedexpressionofsolubleproteinBusingpColdTF.ExpressionofsolubleenzymeproteinB(M.W:~63kDa)wasnotobservedusingeitherpColdDNAIaloneorpColdIco-expressedwithchaperoneproteins,norwithaT7expressionvectorthatincludedothertagsforsolubilization(TrxTag[~12kDa],NusTag[~55kDa],andGSTTag[~26kDa]).

However,whenthepColdTFDNAVectorwasused,thetargetproteinwasobservedatanexpressionlevelmuchhigherthanthatachievedwithothersystemsandtags,andmostoftheexpressedtargetproteinwasobservedinthesolublefraction.(Note:duetothepresenceofvarioustags,targetproteinmolecularweightappearslargerandmorevariablethanitsactualsize).

Insummary,thepColdTFexpressionsystemoffersseveraladvantagesincludingconvenience,highyield,andhighpurity.ThepColdTFsystemissuitableforefficientsolubleproteinexpressioninE.coliofotherwiseintractabletargetproteins.

Figure 1: Expression of Soluble Protein A Using the pCold TF Expression System

pCold TF pColdpCold +

Chaperone T71 2 1 2 1 2 1 2

kDa

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2. Soluble fraction

target protein

co-expressed

trigger factor**


Thedevelopmentofnovelenzymesandproteinsreliesuponanunderstandingoftheirintrinsicstructures.Aprotein’s3-dimensionalstructuredictatesthepositionsofexposedreactivegroupsaswellashiddenhydrophobicresidues,therebydefiningitsbiologicalactivity.Three-dimensionalstructurecanalsoinfluenceprocessessuchasproteintraffickingtoandbetweencellularorganelles.Itiswell-documentedthatseveralhumandiseasesincludingAlzheimer’sDisease,Parkinson’sDisease,andCysticFibrosisariseduetomisfoldedcellularproteins.

In vivo,smallpolypeptidesoftenfoldspontaneouslyintotheircorrectconfiguration.However,longerpolypeptideshaveagreaterlikelihoodoffoldingmoreslowlyandhavegreaterpotentialtoformpartiallyfoldedintermediatestructuresandaggregatesthatarenon-functional.Thetimingofpolypeptidefoldingcanalsoinfluencethesuccessrateofcorrectproteinfolding.Forexample,outcomesmayveryifproteinfoldingisconcomitantwithribosomalsynthesisorifitisdelayeduntiltransportintothecytoplasmortocellularorganelles.

Oneofthelargestproblemsencounteredbyscientistsattemptingtoexpressrecombinantproteinsinbacteriaistheformationofinclusionbodies,insolubleaggregatesofmisfoldedpolypeptidesthatareproducedasthebacteriumquicklysynthesizeslargequantitiesoftheforeignprotein.Thesemisfoldedproteinaggregatesmustbeunfoldedandrefoldedtoassumetheircorrect3-dimensionalstructurebeforefurtherstudyorproductioncanproceed.Asresearcheffortshavecontinuedtoshiftfromtheinvestigationofgenestructuretothestudyofproteinstructureandfunction,theimportanceofstudyingrecombinantproteinshasincreased.Robustmethodsforproducingaccuratelyfoldedstructuresarethereforeessential.

Molecular chaperonesMorethan40yearsago,FerruccioM.RitossafromtheInternationalLaboratoryofGeneticsandBiophysicsinNaples,Italy,firstdiscoveredtheheatshockresponseofcertainproteinsthroughobservationofanewpuffingpatterninDrosophilabuschiisalivaryglandpolytenechromosomes(1).Thispuffingwasindicativeofincreasedgeneexpressionoftheseproteins,whichwerelatertermedheatshockproteins(HSPs).HSPsandrelatedproteins(e.g.DnaK,Hsp40,GrpE;GroEL/S;triggerfactor;prefoldinCCT;SecB;ClpA)arecalled“molecularchaperones”becausetheyhelpensurethatpolypeptidesassumethecorrectconformation.

Oneofthemostimportantfunctionsofchaperonesisinaidingtheprotein-foldingprocess.Becausemolecularchaperonescoevolvedwithpolypeptides—thepresenceofhighlyconservedsequencedataforchaperonesandrepresentationofthesemoleculesineverymajortaxon,includingeukaryotes,eubacteria,archaea,andviruses,suggeststhatchaperonesareancientmolecules—theyprovideforacontrolledcellularmechanismbywhichproteinscanreachtheirmostthermodynamicallystable3-dimensionalconformation.

Chaperonesaccomplishcontrolledproteinfoldingeitherbydirectlybindingtoconserveddomainsinanascentpolypeptideandpreventinginteractionswithotheradjacentproteindomainsorbyproviding,throughtheirown3-dimensionalstructure,aspacethatallowscontrolledpolypeptidefolding(2).Foldingtypicallyoccursviaseveralbindingandreleaseeventswiththepolypeptide,whicharemediatedbythehydrolysisofATP.Butevenwithallofthechaperonemachinerypresentinacell,30%ormoreofallsynthesizedpolypeptidescannotfoldcorrectlytoformfunctionalproteins(3).

Nonetheless,whenthereisneedtorefoldproteinstotheircorrectform,molecularchaperonesareessentialtools.

Refolding strategiesThemostcommonstrategycurrentlyusedtorecoveractiverecombinantproteinin vitrofromisolatedinclusionbodiesconsistsofathree-stepprocess:(1)isolationandwashingoftheinclusionbodies,(2)solubilization(i.e.,denaturationandunfolding)oftheproteinaggregates,and(3)correctrefoldingofthesolubilizedprotein.Thefirsttwoofthesestepstypicallycanbeperformedwithhighefficiency.However,misfoldingandaggregationofthesolubilizedproteinmaycomplicatethelaststep.

Anumberofcommercialkitsareavailableforimprovingtherefoldingconditionsofinclusionbodies,includingFold-It,developedbyHamptonResearch;Pro-MatrixProteinRefoldingKitfromPierce;andNovagen’sProteinRefoldingKit.Theircomponentsincludeadenaturanttosolubilizetheinclusionbodies,oneormoresmall-moleculedetergentstomaintaintheproteininanunfoldedconfigurationandallowrefoldingthroughtransientinteractionswiththeprotein,andvariousbuffersthatdifferinparametersthatinfluencetherefoldingprocess,suchaspH,redoxconcentration,andionicstrength.Thedegreeofsuccessfulrefoldingobtainedbythe“dilutionadditive”strategyofthesekitsdependsonthebufferproperties,aswellasproteinconcentrationandtemperature.

TheRefoldingCAKitfromTakaraBiooffersanovelapproachtothedilutionadditivemethod.Byusinganartificialchaperone—highlypolymerizedcycloamylose(CA)—thekitprovidescorrectrefoldingofaggregatedproteinswithhigheffiency.CAaidsrefoldingthroughitsstructure,asinglehelicalV-amyloseconformationcontainingananhydrophilicchannel-likecavity(4).Inclusioncomplexeswithothermoleculessuchasdetergentscanbeformedinthisspace,preventingproteinaggregation.Incontrasttotheconventionaldilutionadditivemethod,CAsupportsstableinteractionsbetweentheproteinanddetergent,andthenstripsthedetergentfromtheprotein–detergentcomplextoinitiaterefolding(5).Thisscenarioresultsingreaterrefoldingefficiency.CAcanbeusedincombinationwithmanydifferenttypesofdetergentsandiscompatiblewithredoxreagents.Inaddition,CAcaninteractwithpeptidesofmultiplesizes,ishighlysolubleandhasalongshelflifeinaqueoussolution,andaccomplishesprotein

UnfoldingthePotentialofProteins

New refolding technologies are essential for tomorrow’s recombinant proteins ByJobyMarieChesnick

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Figure 1. Comparison of citrate synthase activity following protein refolding using cycloamylose and Tween 40 or Tween 60. Full(100%)activityisachievedforrefoldingofcitratesynthaseusingaslittleas0.6%cycloamylosewithTween60.

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refoldinginashorttime(i.e.,afewhourstoovernight).Figure1showstheamountofcitratesynthaseactivityrecoveredfollowingenzymerefoldingusingCA.Inthisapplication,fullenzymeactivitywasobtainedusing0.6%CAwithTween60detergent(polyoxyethylenesorbitanmonostearate)or1%CAwithTween40(polyoxyethylenesorbitanmonopalmitate).

Analternativein vivomethodforobtainingincreasedsolubleexpressedproteinreliesonchaperone-containingplasmidvectors.Inthiscase,a“team”ofchaperonegenes,suchasDnaK-DnaJ-GrpEorothers,isengineeredintoaplasmidvectorandplacedundercontrolofeitheranaraBorPzt-1promoter,withinductionbyL-arabinoseortetracycline,respectively.Chaperonesarethenco-expressedwiththetargetproteinofinterestinE. coli.Separateexpressionofchaperonesandtargetproteinscanbeaccomplishedifthetargetgeneisplacedunderthecontrolofadifferentpromoter(e.g.,lac).

TheChaperonePlasmidSet,developedbyHSPResearchInstituteandintroducedintothemarketbyTakaraBio,containsfivechaperone-team-containingplasmidsforincreasingyieldsofsolubleforeignproteinsin vivo.TheseplasmidscarryapACYCoriginofreplicationplusachloramphenicolresistancegene,whichallowstheirusewithColE1-typeplasmidscontaininganampicillinresistancegene.Clonesmaybeselectedbasedonpresenceofbothachaperoneplasmidandaplasmidcontainingatargetgeneofinterest.Figure2showsincreasedamountsofsolubletargetprotein(bacterialproteinD),withaconcomitantdecreaseininsolubleprotein,obtainedbyco-expressionofchaperonesfromTakara’spG-KJE8chaperoneplasmidvector.

Forobtaininghighyieldsofrecombinantproteins,TakaraBiooffersplasmidsthatutilizethecold-shockproteinA(cspA)genepromotersystemforgeneexpression.Thissysteminducesproteinexpressionatlowtemperatures(15°C),whichsuppressesthesynthesisofmostotherproteinsandlowerspotentiallydestructivecellularproteaseactivity.Asaresult,upto60%ofallexpressedcellularproteinisthedesiredtarget.Additionally,high-efficiencymetaboliclabelingoftheproteinispossibletofacilitatestructuralanalysis.Large-scalecultureandaffinitypurification(upto400mgofproteinperliterofculture)isachievable,makingcoldshockproteinexpressionsuitableforcommercialapplications.

ThepColdVectorseriesfromTakaraBioallowinsertionofaforeigntargetgeneintoavectorforexpressionusingthecspApromoter.Thecombinationofchaperoneplasmidandcold-shockvectortechnologiesprovidesasystembywhichhighspecificity,high-yieldproductionofsolubleexpressedforeignproteinsisroutinelypossibleataffordablecosts.

Theavailabilityofnewandmodifiedproteinsandenzymesformedicalandbiologicalresearchwilldependtoalargeextentonthedevelopmentofquick,reliable,andcost-effectiverefoldingmethods.

Reprinted with permission from Modern Drug Discovery, July 2004, 7(7), 67.Copyright 2004 American Chemical Society.

References:(1)Ritossa,F.(1962)AnewpuffingpatterninducedbytemperatureshockandDNPin

Drosophila.Experientia18:571-573.

(2)Hartl,F.U.;Hayer-Hartl,M.(2002)Molecularchaperonesinthecytosol:fromnascentchaintofoldedprotein.Science295(5561):1852-1858.

(3)Schubert,U.,etal.(2000)Rapiddegradationofalargefractionofnewlysynthesizedproteinsbyproteasomes.Nature40(6779):770-774.

(4)Machida,S.,etal.(2000)Cycloamyloseasanefficientartificialchaperoneforproteinrefolding.FEBS Letters486(2):131-135.

(5) Daugherty, D.L., et al. (1998) Artificial chaperone-assisted refolding of citratesynthase.J.Biol. Chem.273(51):33961-33971.

Figure 2. Soluble protein production with co-expression of chaperone genes from Takara’s chaperone plasmid vectors with a bacterial gene.Abacterialgenewaspresentatrelativelylowlevelinthesolublefractioninabsenceofco-expressedchaperones,butsolubilitywasenhancedwhenchaperoneswereco-expressed.

Arrowindicatestargetprotein.

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1) What strategies can be used to facilitate production of soluble protein of interest when it is initially expressed in insoluble form?Possiblestrategiesare:

• ChangethetimeatwhichIPTGisaddedtoinducetheexpression.Itmaybenecessarytooptimizethemostidealtimepoint,whichcanrangefromearlytolatelogarithmicphasesoftheculture.

• ReducetheconcentrationofIPTG(downto0.1mM).• ChangethestrainofhostE. coli.• Extracttheculturedcellsbysonicationinabuffercontaining0.1to1%ofdetergent

(forexample,octylglycoside,NP-40,TritonX-100,etc.)

2) How do I select the type of Cold-shock expression vector for SPP to use?TheTranslationEnhancingElement(TEE)facilitatestranslationwhenusingpColdI(SP-4),pColdII(SP-4)andpColdIII(SP-4).ProteinsexpressedusingpColdI(SP-4)andpColdII(SP-4)includeHis-tagsandcanbeaffinity-purifiedwithNicolumns.IfyoudonotdesirethepresenceofanyadditionalaminoacidsequencesattheN-terminusoftheproteinofinterest,thepColdI(SP-4)vectorisrecommended,asitallowscleavageoftheTagsequencewithFactorXa.Alternatively,pColdIV(SP-4)lacksbothTEEandTagsequences.

3) What quantity of the protein of interest will be produced from 1 L of culture?TheexpressionlevelusuallyrangesfromseveralmgtoseveraltensofmgperL,althoughitvariesforeachproteinofinterest.A3-Lculturecantypicalresultinmiligramsofpurifiedprotein,providedtheproteinofinterestcanbedetectedbySDS-PAGEfollowedbyCoomassiebrilliantblue(CBB)staining.IntheMazFco-expressionsystem,expressionofnewendogenoushostproteinsissuppressedexceptfortargetprotein.Thisresultsinhighlyspecificproductionoftargetproteinwithtargetproteincomprisingupto90%oftotalprotein,butmayresultingrowthstressforE.coli itself.AccordinglytheexpressionyieldofatargetproteinmaydecreasecomparedtogeneralexpressionwithpColdDNAs.

SPPSystem™(SingleProteinProductionSystem)

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1) What are suitable competent cells for pCold vectors for protein induction and plasmid storage?Takara’spColdVectorsutilizeanE. colicold-shockgenepromoter.Therefore,mostE.coli strainscanbeusedasahostforproteininductionwiththesevectors.

ThefollowingE. colistrainshavebeentestedandaresuitableforusewiththepColdVectors:

• BL21strains• Rosetta(goodforeukaryoticgenes)• Origami(goodforeukaryoticgenes,butgrowveryslowly)• JM109JM109orDH5αcellsaretypicallyusedtoconstructexpressionplasmidswhichareinsertedwithatargetgene.Note,however,thatwerecommendstoringthepColdVectorsasisolatedplasmidDNA,ratherthanintransformedcells.

2) Where can I obtain sequence information for the pCold Vectors?SequencinginformationforallpColdVectorshasbeendepositedinGenBankandisavailablefromtheNCBIwebsite(http://www.ncbi.nlm.nih.gov/).GenBankAccessionNumbersforeachofthepColdVectorsarelistedbelow:

pCold Vector GenBank Accession No. pColdI AB186388 pColdII AB186389 pColdIII AB186390 pColdIV AB186391 pColdTF AB213654

3) Can you tell me exactly where the cleavage site for Factor Xa is located? Is it at Arg-His?FactorXacleavesattheC-terminalsideoflastaminoacid(Arg)oftherecognitionsequenceIle-Glu-Gly-Arg.Thus,theArg-Hiswillbecleaved.

4) How much Factor Xa would be required to cleave 1 mg of expressed protein?TakaraBioscientistshaveusedFactorXa,RestrictionGrade(Novagen)forcleavage.Cleavageconditionsvaryfromproteintoprotein.Forexample,TakaraBioscientistshaveperformedcleavageusing1UofFactorXafor3µgofa60kDaproteinduringanovernightincubationat4°C.

Besuretooptimizethecleavageconditionsforeachtargetproteinonasmallscalepriortoscalingupreactions.

5) We intend to use pCold III for expression of our protein. Our target gene sequence must contain an ATG start codon so that it can also be used with our PCR primers. However, since the TEE element provided on pCold III also has an ATG start codon, will this situation (i.e. the presence of an ATG start site in both TEE and our target gene sequences) pose a problem for expression?Tousethetranslationenhancingelement(TEE)providedinpColdIII,thetargetgenesequencemustbeinframewiththeTEEsequence.TheATGstartcodonintheTEEisusedforproteinexpression.Thus,usingthisvector,yourexpressedproteinwillbeafusionprotein.ToexpressonlythetargetproteinwithoutTEE,usepColdIVinstead.

WerecommendusingtheNdeIrestrictionsiteintheMCSforfusionofaninsertion(gene)sequencewithastartcodon.

Forexample;

Ifthesequenceofthetargetgeneisthis:

ATG AGC GAT AAA ATT ATT CAC.....

Met-Ser-Asp-Lys-Ile-Ile-His-....

andifpColdIIIcontainingTEEisused,thenfusethegeneintotheNdeIsite(CATATG)asshownbelow,wherethegeneATGstartsiteisunderlined:

Nde I site ...ATG AAT CAC AAA GTG CAT ATG AGC GAT AAA ATT ATT CAC..... Theexpressedproteinwillthenbe:

Met-Asn-His-Lys-Val-His-Met-Ser-Asp-Lys-Ile-Ile-His-...

ForPCRamplificationofthetargetgene,designthePCRforwardprimerconsideringthepointspresentedabove.

6) I recently purchased the pCold I DNA vector and I was wondering what antibody if any could be used with the recombinant proteins produced using this vector?SincemostproteinsproducedusingthepColdVectorswillincludeaHis-tag,TakaraBiorecommendsusingaHis-tagantibody.ClontechoffersthreeHis-tagantibodies:6xHisMonoclonalAntibody(Albumin-Free)(Cat.#631212),6xHNPolyclonalAntibody(Cat.#631213),and6xHismAb-HRPConjugate(Cat.#631210).SeetheClontechRelatedProductssectiononpage31fororderinginformation.

7) Have any of the pCOLD vectors been used for expression in a cell-free system? TakaradoesnothaveinformationaboutuseofpColdVectorsforexpressioninacell-freesystem.However,itisquitepossiblethatgeneralcell-freesystemsarenotsuitableforusewiththepColdVectorssincethesesystemsareusuallyoptimizedforexpressionat37°C.

pColdExpressionVectors


RefoldingCAKit1) What is the difference between the two different sizes of Refolding CA Kits?ThesmallerRefoldingCAKit(Cat.#7350)hasenoughreagentsfor25refoldingreactions.Thisproductissoldasaninitialkitforrefoldingsmalleramountsofproteins(typicallynotmorethan0.24mgproteinperreaction;eachreactionuses~24µlofa10mg/mlinclusionbodysolution)andfortestingrefoldingconditionsusingtheprovideddetergentswiththeCA.Thesmallkitincludesdenaturant(8MGdmCl),4MDTT,4differentsurfactants(1%Tween40,1%Tween60,1%CTAB,1%SB3-14),100mMDL-Cystine,and3%cycloamylose(CA).Notethatureacanalsobeusedwithourkit,althoughitisnotincludedasacomponent.Pleaserefertotheproductmanualfordetailsregardingthekitprotocol.

Thelargekit(Cat.#7351)isintendedforuseafteroptimalrefoldingconditionshavebeendeterminedusingthesmallkit,and/orwhenthereisneedtorefoldlargeramountsofprotein.Thelargekitincludes10-foldmoredenaturant(8MGdmCl)(=20mL)andcycloamylose(CA)(=120mL)thanthesmallkit.Thelargekitdoesnotincludethedetergents,DTT,orDL-Cystine.Thesereagentsareavailableseparatelyfromchemicalreagentsuppliers(e.g.,SigmaAldrich).

2) Could you provide the name of a source for DTT, DL-cysteine and SB3-14 detergent to be used with the Refolding CA Kit? I want to make sure that we purchase exactly the same items that came in the small Refolding CA Kit.Takarausesthefollowingproductsforthiskit:

Component Supplier Catalog #

DTT nakalai 14128-91

Sigma D5545

DL-Cystine nakalai 10316-41

Sigma C8630

SB3-14 Sigma T0807

(Note:SB3-14isregisteredasN-Tetradecyl-N,N-Dimethyl-3-Ammonio-1-PropanesulfonateatSigma)

ProductsfromtheJapanesemanufacturer“nakalai”maynotbeavailableinyourgeographiclocation.However,Sigma’sproducts(listedaboveinparentheses)arecomparableforusewiththiskit.

3) Can disulfide bond-reforming components, such as reduced/oxidized glutathione, be used with Takara’s Refolding CA Kit? Yes,disulfidebond-formingreagentscanbeusedwiththeRefoldingCAKit.NotethatourKitcontainsDL-Cystinefordisulfidebondreformation.Thefollowingreferenceforuseofreduced/oxidizedgluthathionemaybehelpful:

Machida,S.,etal.(2000)Cycloamyloseasanefficientartificialchaperoneforproteinrefolding.FEBS Lett.486(2):131-135.

ThisreferencerecommendsusingGSH:GSSHataratioof5:1.

4) Are all of the detergents removed from solution after addition of cycloamylose (CA)?ThesurfactantsformaprecipitantwithCA,andthemajorityofthesedetergentsarethusremovedafterthefinalcentrifugationstep.However,asmallamountofresidualsurfactantsremaininthefinalrefoldingproteinsolution.

Toremovefreesurfactants,wesuggestusingBioBeads(Bio-Rad).Adda1/5volumeofBioBeadstothefinalrefoldingproteinsolutionandstirthemixturefor2hours.Afterstirring,theBioBeadsareremovedandthesolutionisrecovered.Usingthismethod,morethan95%ofionicsurfactantsandmorethan90%nonionicsurfactantsareremoved.Itis,however,difficulttocompletelyremoveALLtracesofsurfactantsintherefoldingproteinsolution.

5) I do not get a precipitate in the very last step of the protocol when I use either Tween 40 or Tween 60 with the CA, but I do get a precipitate if I use either of the other two detergents. Why am I not getting a precipitate with the Tween 40 or Tween 60?OnrareoccasionsTween40orTween60willsometimesfailtoformaprecipitateduringthelaststep;however,goodrefoldingresultsarestillobtainedeventhoughawhiteprecipitateisnotobserved.Theexactbufferorsaltcompositionandconcentrationmayinfluenceprecipitateformation.However,atpresentthereisnotaclearexplanationastowhyvisibleprecipitateformationmaybevariable.

Ifyoudonotobserveaprecipitate,continuewiththeprocedureregardless.Youshouldperformcentrifugationat15,000rpmfor10minutes,andthencollectthesupernatanttoassesstheproteinactivity.

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1) We are trying to express a protein which contains a few rare codons. This means that we need to express the protein in special strains as Stratagene BL21 Codon plus or Novagen Rosetta. Is there any incompatibility of your Chaperone Plasmids with these kinds of strains? NovagenRosettaandStratageneBL21CodonplusaretheonlyE. colistrainsthatencoderarecodons.Unfortunately,thesestrainsarechloramphenicol-resistant.BecauseTakara’sChaperoneplasmidsallcontainapACYCoriandchloramphenicol-resistancegene,thesestrainscannotbeusedasahostforco-expressionwiththechaperoneplasmids.

2) We have a 64 kDa protein that we have expressed in E. coli. After induction there is a reasonable amount of soluble protein, but most of this protein is not functional, probably because is not properly folded. Can Takara’s Chaperone Plasmids help us to overcome problems associated with expressing a larger protein such as this one? Takarahassuccessfullyfoldeda70kDaproteinusingacombinationofpColdVectors(whichutilizecoldshockexpressiontechnologyforhighyield-highpurityproteinexpression)inconjunctionwithoneofourChaperonePlasmids.

3) After I have used the Chaperone Plasmids for correct folding of my target protein in vivo, can I successfully purify the expressed target protein without co-purification of the chaperone protein? Normally I use a His-tag to purify my expressed proteins, but a His-tag can interact with dnaK. TakaraoftenusesHis-tagstopurifytargetproteinsfoldedin vivousingtheChaperonePlasmids,andhasneverexperiencedchaperoneproteincontamination,suchaswithdnaK,withtargetproteinsthatarepurifiedusingHis-tag/His-bindresin.

FortargetproteinpurificationusingHis-bindresin,besuretouseanappropriatebindingbufferforpurificationwiththeproperconcentrationofNaClandimidazole.

However,sincechaperoneproteinssuchasDnaKorDnaJdotendtonon-specificallybindtotheresin,itmaybesomewhatdifficulttopurifyGST-fusionproteinsbyaffinitycolumnpurification.Inthiscase,werecommendusingbufferthatcontainsATPandMg2+,orusinganATP-agaroseresinforreleaseofproteinfromthecolumn.Forfurtherdetailsoftheseprocedures,pleaserefertothefollowingreferences:

MYu,S.,etal.(1992)InvolvementofthechaperonindnaKintherapiddegradationofamutantproteininEscherichiacoli.EMBO J.11(1):71-77.Zylicz,M.,etal.(1984)PurificationandpropertiesoftheEscherichiacolidnaKreplicationprotein.J. Biol. Chem.259(14):8820-8825.

4) Are restriction maps available for any of the vectors in the chaperone plasmid set?RestrictionmapsforTakara’sChaperonePlasmidsarenotavailable.

However,informationisavailableforthoserestrictionenzymeswhichcuteachplasmidonlyonce.Theseenzymesarelistedbelow.Thenumberin()isthe5’terminalofeachdigestionsite.

• -pKJE7;KpnI(5660),NheI(6606),SacI(4157),ScaI(410),SpeI(5632)• -pG-KJE8;NheI(10608),SacI(4157),ScaI(410),XhoI(8181)• -pGro7;BglII(3929),EcoRI(5462),HindIII(3941),NheI(4880),ScaI(410),

SmaI(3832),XbaI(4040)• -pG-Tf2;BamHI(1979),HindIII(8208),NheI(7268),SalI(4493),ScaI(6271),SmaI

(2786),SpeI(4511),XhoI(1)• -pTf16;BamHI(3323),BglII(1535),HindIII(1523),KpnI(1529),NheI(583),ScaI

(4601),SmaI(2534),XbaI(1424)

5) Has Takara tested any strains of E. coli with these plasmids? Do they have a recommendation for strains that work best with the plasmids?TakarausesmainlyBL21orBL21(DE3)ashoststrainswiththeseplasmids.

AlthoughTakaradoesnothavespecificrecommendationsforE.colistrains,somegeneralizationsmaybemaderegardingtheoptimalcombinationoftargetexpressionvectorandchaperoneteamused.Forexample,thechaperoneteamsofgroES-groELand/ordnaK-dnaJ-grpEmaybemoreusefulthantig(TriggerFactor)tofoldproteinswhichareexpressedusingpETvectors,regardlessofthekindoftargetprotein.

However,ingeneral,werecommendingtestingallfivechaperoneplasmidsforeachtargetproteininordertooptimizeresults.

6) Is DNA sequence information available for the Chaperone Plasmids?Takara’sChaperonePlasmidsarecurrentlyavailableunderalicenseagreementbetweenTAKARABIOINC.andHSPResearchInstitute,Inc.Asaresult,toobtainthesequencesofanyoftheseplasmids,customersarerequiredtocompleteandsubmitaSequenceRequestForm.ThisformisavailableontheChaperonePlasmidSetproductpageatwww.clontech.com/takara.ClickontheorangeboxintheorderingareaatthebottomoftheChaperonePlasmidSetproductpagetoobtaintheform.Uponapprovaloftherequest,thesequenceinformationwillbeforwardedtoyou.

7) Are all chaperone genes encoded on an individual Chaperone Plasmid expressed in the same ratio?ChaperonegeneswhichareunderthecontrolofthesamepromoterinanindividualChaperonePlasmidwillbeexpressedinthesameratio.Forexample,DnaK,DnaJandGrpE,whichareencodedontheplasmidpKJE7,willallbeexpressedinthesameratioincells.ThefollowingreferencesprovideSDS-PAGEdataforchaperoneco-expresssion:

Nishihara,K.,etal.(1998)Chaperonecoexpressionplasmids:differentialandsynergisticrolesofDnaK-DnaJ-GrpEandGroEL-GroESinassistingfoldingofanallergenofJapanesecedarpollen,Cryj2,inEscherichiacoli.Appl. Environ. Microbiol.64(5):1694-1699.Nishihara,K.,etal.(2000)OverexpressionoftriggerfactorpreventsaggregationofrecombinantproteinsinEscherichiacoli.Appl. Environ. Microbiol.66(3):884-889.

8) Is it possible to use the Chaperones Plasmids to aid in vitro translation?Takara’sChaperonePlasmidSetisdesignedtoaidonlyinthefoldingofexpressedproteinsin vivo.WedonothaveanyinformationastowhethertheChaperonePlasmidSetcanbeusedtoaidin vitrotranslation.

ChaperonePlasmidSet


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Product Name Application Description

Pfu Aminopeptidase I •LiberatesN-terminalaminoacidsuptoX-Profromproteinsandpeptides

PfuAminopeptidaseIisathermostableexo-typeaminopeptidase,isolatedfromPyrococcusfuriosusandproducedasarecombinantprotein,whichliberatestheN-terminalaminoacidfromproteinsandpeptides.

Pfu Pyroglutamate Aminopeptidase

•RemovalofpyroglutamicacidsfromtheN-terminalofproteinsandpeptides

•DeblockingofN-terminalpyroglutamatesofproteinsandpeptidesforsequenceanalysisusingEdmandegradation

PfuPyroglutamateAminopeptidaseliberatestheN-terminalpyroglutamicacidfromproteinsandpeptides.Thisenzymemayworkwellwithsomeintact,non-denaturedproteinsandthedenaturationstepmaybeunnecessaryintheseinstances.

Pfu Methionine Aminopeptidase

•LiberatestheN-terminalmethionineresiduesfromproteinsandpeptides

PfuMethionineAminopeptidasespecificallyliberatesonlytheN-terminalmethionineresiduefromMet-X-YwhenXisAla,Gly,Ser,Thr,ProorVal.ThisenzymedoesnotliberatetheN-terminalMetwhentheN-terminussequenceisMet-Met-YorMet-Met-Met-Y.Itisnotactivetowardformyl-methionine.

Pfu N-acetyl Deblocking Aminopeptidase

(Ac-DAP)

•N-Terminaldeblocking

•N-terminalsequenceanalysisofblockedproteinsorpeptides

PfuN-acetylDeblockingAminopeptidase(Ac-DAP)isauniqueexo-typeaminopeptidasethatfirstliberatesblockinggroups,suchasformyl,acetyl,andmyristyl,andthenreleasesthefirstandsubsequentaminoacidsfromproteinsandpeptidesuntilitreachesthefirstX-Probond.

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Arginylendopeptidase •Fragmentationofproteinsandpeptidesrequiredfromprimarystructureanalysis

Arginylendopeptidasecleavespeptidebondsatthecarboxylsideofarginineresiduesfoundinproteinsandpeptides.ArginylendopeptidaseisalsoknownasmousesubmaxillaryproteaseDorasmouseEGFbindingproteinC.

Asparaginylendopeptidase •Fragmentationofproteinsandpeptidesrequiredforprimarystructureanalysis

Asparaginylendopeptidasespecificallycleavespeptidebondsonthecarboxylsideofasparagineresiduesfoundinproteinsandpeptides.Glycosylatedasparagineresiduesarenotcleaved.

Endoproteinase Asp-N •Fragmentationofproteinsandpeptidesrequiredforprimarystructureanalysis

EndoproteinaseAsp-NisametalloproteasethathydrolyzespeptidebondsontheaminosideofAspandCysoxidizedtocysteicacid.Ifcysteineisreducedoralkylated,theenzymewillcleaveonlytheaminosideofAspresidues.

Pfu Protease S •Fragmentationofproteinsandpeptidesrequiredforprimarystructureanalysis

PfuProteaseSisanendo-typeserineproteasewithbroadrecognitionofnativeanddenaturedproteins.Cleavageoccursmainlyonthecarboxysideofpeptidebondsofhydrophobicaminoacidresidues.

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Calpastatin •Calpainproteaseinhibitor

Calpastatinisanendogenousproteaseinhibitorthatactsspecificallyoncalpaincalcium-dependentcysteineprotease.Itconsistsoffourrepetitivesequencesof120to140aminoacidresidues(domainsI,II,andIV),andanN-terminalnon-homologoussequence(L).TheproductconsistsofhighlypurifiedrecombinanthumancalpastatindomainI.

Takara offers a wide variety of Protein Fragmentation products as well as N-terminal deblocking and sequence determination and C-terminal sequence determination products. The fragmentation products are used for analysis of the primary structure of proteins and peptides.

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PrimeSTAR®GXLDNAPolymerase

PrimeSTAR® GXL DNA Polymerase R050A 250 Units PrimeSTAR® GXL DNA Polymerase R050B 1,000 Units

Features• Highest Processivity: among commercially available high-fidelity DNA

polymerases• Antibody-Mediated Hot-Start Formulation• Proven Performance: as reported in peer-reviewed literature

DescriptionCapableofoutstandingperformanceforbothroutinehigh-fidelityPCRandchallengingtemplates or reaction conditions, PrimeSTAR GXL DNA Polymerase is the most robusthigh-fidelityPCRenzymecommerciallyavailable.Itprovideshighyield,highspecificity,and high accuracy for not only standard reactions, but also excels in PCR with GC-richtemplates,inthepresenceofeXcesstemplate,andforamplificationofLongproductsup

to30kb(GXL).SimplifyyourPCRandsavetimebyrelyingononeenzymesystemworksregardlessofconditions,withminimaloptimizationrequired.PrimeSTAR GXL DNA Polymerase includes a modified PrimeSTAR HS enzyme and anadditional elongation factor which in combination provide unsurpassed processivity.PrimeSTARGXLDNAPolymerasehasoutstandingperformanceinreactionscontainingexcess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibitsPCR amplification by conventional polymerases because the amount of effectivepolymerase available is limited by nonspecific binding. The superior processivityof PrimeSTAR GXL DNA Polymerase prevents such inhibition by excess nucleicacid, resulting in a much higher success rate for PCR with minimal optimization ofconditions required. Furthermore, the antibody-mediated hot start formulationprevents false initiation events during the reaction assembly due to mispriming andprimerdigestion.

Applications• Successful, Robust, High-Yield PCR regardless of Conditions• Long PCR: Amplify products up to 30 kb (human genomic DNA), 40 kb

(lambda DNA), or 13.5 kb (human cDNA)• Use on Standard and Challenging Templates alike: Outstanding

performance on GC-rich or AT-rich templates and targets containing repeats

• Can be used with Samples Containing Excess Nucleic Acid: Tolerates a wide range of template quantity, including high levels of template that inhibit other high-fidelity DNA polymerases

• Next Generation Sequencing (NGS) studies involving deep sequencing of the same region in many samples

Hig

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PrimeSTAR®MaxDNAPolymerase PrimeSTAR® MAX DNA Polymerase R045A 100 Rxns PrimeSTAR® MAX DNA Polymerase R045B 400 Rxns

Features• Highest Fidelity: of any commercially available PCR polymerase• Fastest Extension Speed: means less time required for PCR cycles• Convenient Premix: assemble reactions in less time• Antibody-Mediated Hot-Start Formulation• Proven Performance: as reported in peer-reviewed literature

DescriptionPrimeSTAR Max DNA Polymerase is a unique high-performance DNA polymerase forPCR. PrimeSTAR Max DNA Polymerase has the highest fidelity and fastest extensionspeed of any commercially available enzyme, along with extremely high sensitivity,processivity,andspecificity.Itincludesanelongationfactortoprovideefficientprimingandextension,greatlyreducingthetimerequiredforannealingandextensionsteps.Asaresult,PrimeSTARMaxDNAPolymerasecanbeusedforexceptionallyfasthigh-speed

PCR. Since it is formulated as a premix that supports hot-start PCR, it's also excellentfor high-throughput experiments. When you need fast reaction times and/or highlyaccurate amplification for cloning and expression, structural studies, or evolutionaryanalyses,PrimeSTARMaxDNAPolymeraseistheenzymeofchoice.PrimeSTAR Max DNA Polymerase is suitable for reactions occurring in the presenceof excess nucleic acid. Such extraneous DNA in a reaction mix ordinarily inhibits PCRamplification when using conventional polymerases because the amount of effectivepolymerase available is limited by nonspecific binding. The superior processivityof PrimeSTAR Max DNA Polymerase prevents such inhibition by excess nucleic acid,resultinginamuchhighersuccessrateforPCRwithminimaloptimizationofconditionsrequired. Furthermore, the antibody-mediated hot start formulation prevents falseinitiationeventsduringthereactionassemblyduetomisprimingandprimerdigestion.Since PrimeSTAR Max DNA Polymerase is configured as a 2-fold premix containingreactionbufferanddNTPmixture, itallowsrapidpreparationofreactionsandisusefulforhigh-throughputapplications.

Applications• Use Whenever Accuracy and Fidelity is Critical• Cloning and Expression Studies• Structure-Function Studies• Analyses that involve Evolutionary Inferences (SNP analyses, evolu-

tionary development experiments, etc.)• Whenever Fast PCR Cycles are Needed, such as High-Throughput

Studies

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PrimeSTAR® GXL Company I Company TM1234MM1234M1234M

Comparison of Amplification of GC-rich targets using PrimeSTAR GXL and other com-mercially available high-fidelity DNA polymerases.ExcellentresultswereachievedusingPrimeSTAR®GXLDNAPolymerasewithoutrequiringspecialbuffersorreactionconditions.Template: HumangenomicDNA(100ng/50μlreaction)1.APOEgene746bp(GCcontent74%) 2.TGFβ1gene2005bp(GCcontent69%)

Template:T.thermophilusHB8genomicDNA(10ng/50μlreaction)3.2029bp(GCcontent74%)4.4988bp(GCcontent74%)

PrimeSTAR® Max Company T Company S M12345678MM12345678M12345678

Good amplification was observed for products up to 6 kb using an extension time of 10 sec. with PrimeSTAR Max.

Template: λDNA(1ng/50μlreaction)

Target size: 0.5kb(lane1),1kb(lane2),2kb(lane3),4kb(lane

4),6kb(lane5),8kb(lane6),10kb(lane7),12kb(lane8)

Extension time: 10sec


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Ordering Information

Takara has been manufacturing high quality restriction enzymes for over 30 years and offers more than 90 restriction enzymes to meet your cloning needs.

Visit our web site at www.clontech.com/takara

to view these and other products.

Product Name Product No. Quantity Ligation T4 DNA Ligase 2011A 12,500 U (100 Weiss units) DNA Ligation Kit, Mighty Mix 6023 1 kit (75-150 rxns) DNA Ligation Kit, Version 2.1 6022 50 Rxns Cloning Vectors pBR322 DNA 3050 25 µg pUC18 DNA 3218 25 µg pUC19 DNA 3219 25 µg pUC118 DNA 3318 25 µg pUC119 DNA 3319 25 µg Protein Sequencing and Analysis Pfu Pyroglutamate Aminopeptidase 7334 10 mU Pfu Methionine Aminopeptidase 7335 20 mU Pfu Aminopeptidase 7336 0.5 mg Pfu N-acetyl Deblocking Aminopeptidase 7340 50 µg Arginylendopeptidase 7308 0.5 mg Asparaginylendopeptidase 7319 0.2 mU Endoproteinase Asp-N 7329 2 µg Pfu Protease S 7339 500 U Electrophoresis Mupid®-exU Electrophoresis System AD140 1 Unit Mupid®-2plus Electrophoresis System AD110 1 Unit Mupid® One Electrophoresis System AD160 1 Unit Power Supply for Mupid®-2Plus AD111 1 Unit Retroviral Transduction RetroNectin® Recombinant Human Fibronectin Fragment T100A 0.5 mg RetroNectin® Recombinant Human Fibronectin Fragment T100B 2.5 mg RetroNectin® Precoated Dish T110A 10 Dishes Ladders 1 kb DNA Ladder (Dye Plus) 3426A 100 Rxns 100 kb DNA Ladder (Dye Plus) 3422A 100 Rxns 20 bp DNA Ladder (Dye Plus) 3420A 100 Rxns 200 bp DNA Ladder (Dye Plus) 3423A 100 Rxns 250 bp DNA Ladder (Dye Plus) 3424A 100 Rxns 500 bp DNA Ladder (Dye Plus) 3425A 100 Rxns High Fidelity PCR Enzymes PrimeSTAR® GXL DNA Polymerase R050A 250 Units PrimeSTAR® GXL DNA Polymerase R050B 1000 Units PrimeSTAR® Max DNA Polymerase R045A 100 Rxns PrimeSTAR® Max DNA Polymerase R045B 400 Rxns Protease Inhibition Calpastatin 7316 3 mg


Product Name Product No. Quantity TALON Prepacked HisTALON Gravity Columns 635655 5 columns HisTALON Gravity Columns Purification Kit 635654 20 purifications TALON Single Step Columns (20 ml) 635632 10 columns HisTALON Superflow Cartridges (5 x 1 ml) 635650 5 cartridges HisTALON Superflow Cartridge (1 x 5 ml) 635683 1 cartridge HisTALON Superflow Cartridges (5 x 5 ml) 635682 5 cartridges HisTALON Superflow Cartridge Purification Kit (5 x 1 ml) 635649 20 purifications HisTALON Superflow Cartridge Purification Kit (5 x 5 ml) 635681 5 purifications His60 Ni Superflow Resin His60 Ni Superflow 635659 10 ml 635660 25 ml 635661 4 x 25 ml 635662 250 ml 635663 2 x 250 ml 635664 4 x 250 ml His60 Ni Prepacked His60 Ni Gravity Columns 635657 5 columns His60 Ni Gravity Columns Purification Kit 635658 20 purifications His60 Ni Superflow Cartridges (5 x 1 ml) 635675 5 cartridges His60 Ni Superflow Cartridge (1 x 5 ml) 635680 1 cartridge His60 Ni Superflow Cartridges (5 x 5 ml) 635679 5 cartridges His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml) 635674 20 purifications His60 Ni Superflow Cartridge Purification Kit (5 x 5 ml) 635678 5 purifications In-Fusion® HD Cloning System Liquid Kits In-Fusion® HD Cloning System 639645 10 Rxns 639646 50 Rxns 639647 100 Rxns 639692 96 Rxns In-Fusion® HD Cloning System CE 639636 10 Rxns 639637 50 Rxns 639638 100 Rxns 639693 96 Rxns Antibodies c-Myc Monoclonal Antibody 631206 200 μg HA-Tag Polyclonal Antibody 631207 100 μg c-Myc Monoclonal Antibody-Agarose Beads 631208 1 ml 6xHis mAb-HRP Conjugate 631210 100 μl 6xHis Monoclonal Antibody (Albumin-Free) 631212 200 μg 6xHN Polyclonal Antibody 631213 200 μl In-Fusion® HD Cloning System EcoDry™ Kits In-Fusion® HD EcoDry™ Cloning System 639684 8 Rxns 639685 24 Rxns 639686 48 Rxns 639688 96 Rxns Protease Inhibitor ProteoGuard EDTA-Free Protease Inhibitor Cocktail 635672 100 μL 635673 10 x 100 μL

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ClontechRelatedProducts

Ordering Information

TB 633353 Dist

Takara Product OfferingTake advantage of Takara’s broad product portfolio and expert manufacturing capabilities to increase reliability and reproducibility, and to reduce your costs. Takara also offers custom, bulk and OEM services.

PCR Polymerases and Reagents

• Polymerases

• Real-Time (qPCR) reagents

• Reverse transcriptases

• Primers and buffers

Molecular Biology

• Restriction and modifying enzymes

• Cloning vectors

• DNA/RNA ladders and MW markers

• Electrophoresis salts and buffers

Cellular Biology

• Antibodies

• EIA kits

Notice to Purchaser. Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product’s web page at http://www.clontech.com/takara. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. Unless otherwise specified, other trade names are also the trademarks or registered trademarks of various companies.

Protein Research

• Protease Inhibition

• Protein Folding & Expression

• Protein Sequencing and Analysis

Glycobiology

• Glycobiology Enzymes and Reagents

• Glycobiology Kits

TAKARA BIO INC.

www.clontech.com/takara

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