Application Manual QuickFold

7/29/2019 Application Manual QuickFold

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Protein Refolding Kit

Athena Enzyme Systems

888-892-8408

www.athenaes.com

Application Manua

V 2.1

The QuickFold Protein Refolding Kit

employs a fractional factorial matrix

design that allows the researcher to

screen specific protein in 15 different

buffers, examine a wider range of

conditions and pinpoint the critical

factors for refolding their protein

within a single experiment.


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(MD): 410-455-6319

(USA): 888-892-8408

: 410-455-1155

[email protected]

a division of Athena Environmental Sciences, Inc.

1450 South Rolling Road

Baltimore, MD 21227

USA

thena Enzyme Systems

Application Manual

V. 2.1

Athena Environmental Sciences, Inc.

QuickFold Protein Refolding Kit is a trademark of Athena Envi-ronmental Sciences, Inc. Athena Enzyme Systems is a division andtrademark of Athena Environmental Sciences, Inc. AthenaES is atrademark of Athena Environmental Sciences, Inc. and the AthenaESlogo is a registered logo of Athena Environmental Sciences, Inc.

Protein Refolding Kit


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Introduction 3Principle of the Kit 4

Kit Components 6

Protocols:

Preparation 7

Task 1: Screening for Basic Buffer Composition 8

Task 2: Optimation of the Buffer Composition 11

Supplemental Protocols:

Purification of Inclusion Bodies 13

Methods for Increasing Soluble Protein Accumulation 14Increased Soluble Protein Accumulation Using Chaperone Induction 17

References 19

Product Warranty 20

Product Limitations 20

Ordering Information 21

QuickFold Protein Refolding Kit

Application Manual

Table of Contents


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QuickFold Protein Refolding Kit Application Manua

3

Introduction

For more than 20 years . coli has proved to be a reliable host for the production of heterologousproteins. The well defined genetics, readily available host-vector systems, and established

methods has made . coli the first choice for the expression of recombinant proteins. Despitethe history of successes, the expression of heterologous proteins via the production of solublefunctional protein remains unpredictable. Frequently, the over expression of a protein in . coliresults in the formation of insoluble inclusion bodies.

The reasons for inclusion body formation are not fully known. Since translation is a slowerprocess than protein folding, it is likely that the misfolding of translation intermediates playssome role. Posranslational modification, such as glycosylation and lyposylation, are knownto affect the secondary structure of proteins. In bacteria, these modifications are mostly absent.Further, the chemical environment in which translation occurs in the eukaryotic cell is differentthan that of the bacterial cell. Each of these factors contributes to varying degrees to how thenascent polypeptide folds, or in the case of recombinant protein expression, misfolds. 1

Several approaches have been used to mitigate misfolding during the over expression ofproteins in . coli. These include: 1) fusion of the target protein with a more soluble partner,typically a bacterial protein; 2) co-expression of folding catalysts and chaperones; 3) expressionunder culture conditions which reduce the translation rates or affect the intracellularenvironment; and 4) modification of the protein sequence. Each approach has advantages anddisadvantages which must be weighed in light of the intended end-use of the target protein.Further, not all proteins respond favorably to any given approach. Again, which approach is

best suited to a given protein must be determined empirically and success in producing andrecovering soluble active protein is not guaranteed.

Both a bane and blessing, the formation of inclusion bodies renders the expressed protein

unusable. The purification of a protein as an inclusion body is relatively simple, easily scalablefor commercial applications and in many cases can stabilize the protein until a suffi cientdegree of purity is obtained. The challenge is that the protein must then be recovered from theinsoluble particle. The recovery of soluble active protein from purified inclusion bodies requiresthe denaturation of the polypeptide and then its refolding to an active form. Many examplesof proteins recovered from inclusion bodies are well known and used for both commercial andacademic applications. There are well established methods for purifying inclusion bodies andsolubilizing the aggregated protein by denaturation. There is, however, no reliable method forpredicting the conditions needed to refold the protein. Thus, the identification of the conditionsneeded to properly refold the protein remains an empirical science. The purpose of theQuickFold Protein Refolding Screening Kit is to help simplify the process of identifying the

buffer composition and method which is best suited for the refolding of any given protein.

Introduction


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Principle of the Kit


he information for protein folding is coded in the linear sequence of theo ypepti e.2 Wit rare exception eac protein can e enature an re o e into

native active state under the right conditions. However, predicting the foldingathway for any given protein is a daunting challenge. For a 100 residue polypeptideere are 9100 accessi e con rmations. I eac con ormationa searc requires 10-15

econds to complete it would take approximately 2.9 x 1079 years to examine eachossible configuration. This Levinthal paradox is resolved during protein foldingy t e progressive sta i ization o interme iate states. Pro uctive partia y o eonfirmations are retained while non-productive folds are rearranged. The keyppears to e t e cooperative ormation o sta e native- i e secon ary structureshich serve to nucleate the process. In practical terms elucidating the foldingathway for any given protein requires painstaking analysis and significant technicalapa i ities. Unti a more t oroug un erstan ing o t e re ations ip etween

rimary protein sequence and structure is developed and the tools become availableor in silico prediction of protein structure, the best available method for determining

e con itions or protein o ing remains empirica testing.

he parameter affecting protein refolding has been extensively reviewed.3,4,5 Theey to success u y re o ing a protein is to prevent o -pat way pro ucts romccumulating. These unwanted species form aggregates, a process which can be self-

nuc eating, resu ting in poor recoveries o proper y o e proteins. Interme iatesith hydrophobic patches which are exposed to solvent are believed to play a

ignificant role in the formation of off-pathway products. Thus, to avoid off-pathwayro ucts t e main tactic is a continuous or iscontinuous u er exc ange w ere t e

enaturation buffer is designed to minimize these offpathway products.

e o ing o proteins in so ution is a ecte y a num er o p ysioc emicaarameters. These parameters include: Ionic strength, pH, temperature, oxidationtate and protein concentration as well as the presence of hydrophobic, polar,

aotropic agents an ot er proteins. A compre ensive ist is given y C ar 4. T us,he first step to develop a method for refolding proteins purified from inclusion

bodies is to determine the composition of the refolding solution. The QuickFoldProtein Refolding Screening Kit contains 15 different buffer compositions which

ermit the rapid identification of the factors which have a major effect on proteino ing. From t is in ormation experiments can e per orme to etermine t e

ptimum buffer formulation.

Five i erent tec niques are emp oye to exc ange t e enaturant u er withe refolding buffer including dilution, dialysis, diafiltration, gel filtration andmmobilization on a solid support. For screening purposes and, in some cases, smallo mo erate-sca e pro uction, i ution is t e simp est approac . Its o vious raw acs that this technique leads to dilute protein solutions that would subsequently have to

be concentrated; with larger production volumes it would become cumbersome.

4


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T e ot er u er exc ange tec niques are u y sca a e to commercia pro uction ancan be performed under higher protein concentrations. Care must be taken to definet e con itions w ic prevent aggregation un er ig protein concentrations. Severavariations on the basic theme of buffer exchange have been noted for various proteins.For example, a temperature leap in which the target protein is refolded at lowtemperature o owe y a rapi increase in temperature to comp ete t e process asbeen applied to the refolding of carbonic anhydrase II.6 During the low temperatureincubation, folding intermediates which do not aggregate accumulate and upon arapi temperature increase t e na pro uct is orme wit minima mis o ing.Another approach is to expose the protein to intermediate denaturant concentrationsthat prevent the formation of aggregates but allow refolding to occur. This can be

one y rapi i ution o owe y s ow ia ysis into t e na u er (examp e:lysozyme) or by gradually removing the denaturant by dilution during dialysis(examp e: immunog o u in G).7 A genera ru e is t at i a protein orms aggregates atintermediate concentrations of denaturant, that a fast or slow dilution of denaturedprotein into renaturation buffer is best. If the protein does not form aggregates atinterme iate enaturant concentrations, t en s ow ia ysis wit a gra ua remova othe denaturant is best.

5


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Kit ComponentsQuickFold Protein Refolding Kit Components

omponent Amount atalog Number Component Amount atalog Number

Buffer 1 11 mL 0612-1, 0613-1 Buffer 10 11 mL 0612-10, 0613-10



Buffer 4 11 mL - , - Buffer 13 11 mL - , -



Buffer 7 11 mL - , - DTT 1 mL Stock

Buffer 8 11 mL 0612-8, 0613-8 Glutathione, reduced 1 mL Stock

Buffer 9 11 mL 0612-9, 0613-9 Glutathione, oxidized 1 mL Stock

QuickFold Protein Refolding Buffer Formulation

omponent Formulation after Preparation (Addition of DTT, GSH, and GSSH) DTT GSH/GSSH

Buffer 1 50 mM MES pH 6.0, 9.6 mM NaCl, 0.4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.75 M GuanidineHCl, 0.5% Triton X-100, 1 mM DTT

Buffer 250 mM MES pH 6.0, 9.6 mM NaCl, 0.4 mM KCl, 2 mM MgCl , 2 mM CaCl , 0.5 M arginine, 0.05%

polyethylene glycol 3,550, 1 mM GSH, 0.1 mM GSSH

Buffer 350 mM MES pH 6.0, 9.6 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 0.4 M sucrose, 0.75 M Guanidine

HCl, 0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 1 mM DTT

Buffer 450 mM MES pH 6.0, 240 mM NaCl, 10 mM KCl, 2 mM MgCl

22 mM CaCl

20.5 M arginine, 0.5%

Triton X-100, 1 mM GSH, 0.1 mM GSSH

Buffer 550 mM MES pH 6.0, 240 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.4 M sucrose, 0.75 M Guanidine

HCl, 1 mM DTT

Buffer 650 mM MES pH 6.0, 240 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.4 M sucrose,

0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 1 mM GSH, 0.1 mM GSSH

Buffer 750 mM MES pH 6.0, 240 mM NaCl, 10 mM KCl, 2 mM MgCl 2 mM CaCl 0.75 M Guanidine

HCl, 0.05% polyethylene glycol 3,550, 1 mM DTT

Buffer 850 mM Tris-Cl pH 8.5, 9.6 mM NaCl, 0.4 mM KCl, 2 mM MgCl 2 mM CaCl 0.4 M sucrose, 0.5%

Triton X-100, 0.05% polyethylene glycol 3,550, 1 mM GSH, 0.1 mM GSSH

Buffer 950 mM Tris-Cl pH 8.5, 9.6 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.75 M Guanidine

HCl, 0.05% polyethylene glycol 3,550, 1 mM DTT

Buffer 1050 mM Tris-Cl pH 8.5, 9.6 mM NaCl, 0.4 mM KCl, 2 mM MgCl

22 mM CaCl

20.5 M arginine, 0.4

M sucrose, 0.75 M Guanidine HCl, 1 mM GSH, 0.1 mM GSSH

Buffer 11 50 mM Tris-Cl pH 8.5, 9.6 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT

Buffer 1250 mM Tris-Cl pH 8.5, 240 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.05% polyethylene glycol 3,550,

1 mM GSH, 0.1 mM GSSH

Buffer 1350 mM Tris-Cl pH 8.5, 240 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.75 M Guanidine

HCl, 0.5% Triton X-100, 1 mM DTT

Buffer 14

50 mM Tris-Cl pH 8.5, 240 mM NaCl, 10 mM KCl, 2 mM MgCl , 2 mM CaCl , 0.5 M arginine, 0.4

M sucrose, 0.75 M Guanidine HCl, 0.5% Triton X-100, 0.05% polyethylene glycol 3,550, 1 mM

GSH, 0.1 mM GSSH

Buffer 1550 mM Tris-Cl pH 8.5, 240 mM NaCl, 10 mM KCl, 2 mM MgCl

2, 2 mM CaCl

2, 0.4 M sucrose, 1

mM DTT

Kit Components

6


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1.

2.

3.

Protocols

Preparation: DTT, GHS, GSSH

Dissolve contents of the DTT vial in 1 mL of deionized water. Store at 4C.Dissolve contents of the Glutathione, reduced (GSH) vial in 1 mL of deionizedwater. Store at -20C.

Dissolve contents of the Glutathione, oxidized (GSSH) vial in 1 mL of deionizedwater. Store at -20C.

1.

2.

3.

Preparation: Buffers

Add 1 l DTT solution to 950 l of the respective buffer as follows:Bu ers: 1, 3, 5, 7, 9, 11, 13, & 15

Add 1 l GSH solution to 950 l of the respective buffer as follows:Bu ers: 2, 4, 6, 8, 10, 12, & 14

Add 1 l GSSH solution to 950 l of the respective buffer as follows:Bu ers: 2, 4, 6, 8, 10, 12, & 14

Protocols

Preparation

Task 1: Screening for the Basic Buffer Composition

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Purify the protein as inclusion bodies and solubilize in neutral buffered 8M urea.Supplement the buffer with DTT as required by the protein. Supplemental Protocol1 gives met o s or puri ying an preparing inc usion o ies.

Adjust the protein concentration to 1 mg/ml.

Dispense 950 l of each buffer into each of 15 1.5 ml polypropylene microfugeubes. Dispense 950 l of the denaturation buffer into tube number 16.

Slowly add 50 l of the protein solution to each tube while vortexing the solutiongently.

Incubate at 4C or 22C for 1 hour.

Micro uge or 5 min.

Carefully pipet the liquid into a clean tube. This should contain refolded, solublerotein. Reserve t e pe et.

Assess successful refolding as follows:

By Functional Assay:

It is best to perform a functional assay to determine if any active proteins present.

By Immunoassay:

Per orm an immuno ot, s ot/ ot ot or microp ate assay. App y 2.0g of protein per well. It is important for slot/dot blot and microplatessays that appropriate controls are used to ensure that the signal

taine is ue to t e target protein an not non-speci c anti o ybinding. Extracts prepared from an isogenic parent strain is best.

Use a quantitative ensitometery scan to etermine t e re ative amountf protein recovered.

By SDS-PAGE:

Mix 40 l of the soluble fraction with 10 l 5x SDS-PAGE Loading Dye.

7.

8.

8.1.

8.2.

8.3.

8.1.1.

8.2.1.

8.2.2.

8.3.1.

8

Protocols



Because t ere are a myria o c emica an p ysica con itions w ic can a ectprotein refolding, the systematic survey of each factor would be a time consuming

process. The screen employed by this kit is based on a fractional factorialexperimental design. This allows the researcher to determine the critical factorsaffecting protein refolding and thus to quickly define a suitable refolding regime.

1.

2.

3.

4.

5.

6.


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Heat at 100C or 5 min.

Load 10-20 l per lane of a gel. This will give 0.4 to 0.8 mg of the targetrotein per lane. A Tris-glycine SDS gradient acrylamide gel of 4-20%

s recommended.

Aer electrophoresis, stain with Coomassie Blue.

Success u re o ing is evi ence y t e presence o t e target proteinn the liquid fraction.

Per orm a quantitative ensitometery scan to etermine t e re ativemount of protein recovered.

By Size Exc usion C romatograp y:

Inject 0.1 ml of the solution containing the refolded protein into aa i rate 300 x 7.8 ID mm SEC co umn an c romatograp . (Amm resin with a 300- pore size is recommended. See Goodingnd Freiser, 19918 and Engelhardt, 19919 for a general discussion ofna ytica SEC on proteins.)

Misfolded or aggregated protein will have a different retention timehan the correctly folded protein.

Interpret t e ata.

Successful refolding is achieved when >30% of the input protein or activityis recovere in t e so u e raction. (Yie = Amount o proper y o eprotein recovered/Amount of protein input.)

Determine t e actors w ic are aving a major e ect on protein re o ing.

Prepare a spreadsheet with 15 rows corresponding to reactions 1 to 15n 14 co umns correspon ing to eac o t e actors teste (13) anhe solutions as shown in the figure on the next page.

Enter t e va ue (i.e., enzyme activity, mass, etc.) o taine or eac

olution into each cell in the row. For any given solution each factorill have the same value entered. (Note: Numeric descriptors for

ua itative assessments wi a so wor , ut wit ess accuracy.)

Calculate the sum of protein recovered for each factor when the factoras present in the solution. SumPresent.

Ca cu ate t e sum o protein recovere or eac actor w en t e actoras absent from the solution. SumAbsent

9.

8.4.

9.1.

9.2.

9.2.1.

9.2.2.

9.2.3.

9.2.4.

Protocols


9

8.4.1.

8.4.2.

8.3.2.

8.3.3.

8.3.4.

8.3.5.

8.3.6.

9.1.

9.2.


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9.2.6.1.

9.2.6.2.

9.2.6.3.

Ca cu ate t e i erence etween t e Present an A sent an ivi eby 7.5 for each factor. Relative Effect = Sum resent - SumAbsent / 7.5.

Compare the Relative Effect numbers obtained.

A positive num er in icates a positive e ect on re o ing.

A negative number indicates no effect on refolding.

he larger the positive number the greater the effect of thegiven factor.

Apply this same set of calculations to any other parameters used toest protein refolding such as temperature, protein concentration, etc.

9.2.5.

9.2.6.

9.2.7.

Protocols


QuickFold Protein Refolding Buffer Formulation

Solution pH 6.0 pH 8.5 NaCl/KCl Mg/Ca .... .... GSH/GSSH

.... ....

.... ....

- - -

14 .... ....

15 .... ....

SumPresent

.... ....

SumAbsent

.... ....

Relative Effect = Sumresent

- SumAbsent

/ 7.5 .... ....

Table 1. Example analysis table used to determine the factors critical to protein refolding. Anxcel spreadsheet is available for download at www.athenaes.com.

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Task 2: Optimization of the Buffer Composition

Once t e critica parameters ave een i enti e , t e re o ing con itions s ou eoptimized. The extent to which the optimal conditions must be defined depends on

the intended use of the protein and whether or not additional purification steps areneeded. The following is a general scheme for optimizing the refolding procedures.

Determine the optimal buffer.

Test or protein re o ing as in Tas 1 step 8 using t ree eve s o eac o t ecritical factors. Select the maximum, minimum and median values for eachfactor. The experimental design can be to vary each factor individually oremp oy a statistica esign wit a t ree eve ana ysis.10

Refine the optimum factor levels by titering the factor levels within the

ranges e ne in step 1.1.

For preparations with more than 20 mg of the target protein, test forre o ing at ig protein concentrations, i.e., >1 mg/m . T is can e oneusing the dilution technique or dialysis.

Sca e t e re o ing to t e esire eve .

For 1-20 mg protein, the dilution method will suffi ce. Aererforming the refolding step, concentrate the protein by

ultrafiltration or chromatography and exchange the buffer to one

uita e or t e inten e use o t e protein. It may e possi e to usinghe dilution method for up to 100 mg of protein if the refolding canccur at protein concentrations above 0.1 mg/ml.

For refolding more than 20 mg of protein, an alternative methodhould be employed which does not dilute the protein. The followings a simp e approac w ic most a oratories can rea i y usend is generally applicable. Alternative techniques are discussedlsewhere.3,4,5

Prepare 2 liters of Solubilization Buffer (see Task 3 step 1.3)

Prepare 4 iters o Re o ing Bu er (as etermine a ove).

Fill a 3,500 NMCO dialysis membrane with the denaturedrotein solution. The protein concentration should be at theaximum possible as determined during the refolding

ptimization process.

1.

1.1.

1.2.

1.3.

1.4.

1.4.1.

1.4.2.

1.4.2.1.

1.4.2.2.

1.4.2.3.

Protocols


11


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Dia yze against 2 iters o So u i ization Bu er or 2 ours.

Continuously add the Refolding Buffer at the rate of 1 ml/minhile removing the Solubilization Buffer at the same rate.

A er 48 ours, remove t e ia ysis ag rom t e so ution anialyze against 2 liters Refolding Buffer at 4C for 2-4 hours.

Dia yze against t e u er nee e or t e inten e use o t erotein.

Remove any precipitate materia y centri ugation at20,000 xg for 20 min. at 4C.

1.4.2.4.

1.4.2.5.

1.4.2.6.

1.4.2.7.

1.4.2.8.

Protocols


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2.

Supplemental Protocol 1: Purification of Inclusion Bodies

T ere are a myria o approac es or puri ying inc usion o ies. T e most commontechnique for bench-scale applications is centrifugation, with diafiltration andcontinuous ow centri ugation eing use or commercia -sca e operations. It is mostoen best to purify the inclusion bodies as insoluble products and the dissolve themin denaturant before refolding. This will remove unwanted contaminants, especiallyproteases. Once t e inc usion o ies are re ative y pure, t ey are so u ize wit6 M guanidine HCl or 8 M Urea. The solubilization is protein-dependent and theconditions needed with regard to the concentration of denaturant, ratio of denaturantto protein, pH, ionic strengt , time o exposure to enaturant, temperature, re oxagents or derivatization of thiol groups should be determined empirically. Themethod below is a general scheme which will work for most proteins. Alternative

approaches and more extensive discussions on inclusion body purification can befound in the literature. ,4,5,11

Materia s:

Cell pellet of the strain in which the target protein was expressed.

Wash Buffer: 4 M Urea, 0.5 M NaCl, 1 mM EDTA, 1 mg/ml deoxycholate,50 mM Tris-Cl pH 8.0. (Note: The optimal urea and salt concentrationshould be determined in a pilot experiment. This is done by resuspendingthe insoluble material from an extract in buffer with different levels of

urea. Se ect t e ig est urea an sa t concentration t at oes not so u i izethe target protein. A nonionic detergent may be included in the buffer toimprove the purity. Its optimal concentration should be determined as fort e urea an sa t.)

Solubilization Buffer: 6 M Guanidine-HCl (or 8 M Urea), 50 mM Tris-ClpH 8.0, 10 mM DTT.

Methods

Prepare a cell-free extract and clarify by centrifugation at 20,000 x g for30 min at 4C.

Wash the pellet twice with 5 ml/g Wash Buffer. Centrifuge at 20,000 x g for15 min at 15C.

Suspend the pellet in Solubilization Buffer at 2 ml/g. Heat at 50C for 10-20min to aci itate isso ution.

Clarify the solution by centrifuging at 20,000 x g for 30 min. Reserve thesupernatant.

1.

1.1.

1.2.

1.3.

2.1.

2.2.

2.3.

2.4.

13

Supplemental Protocols

Purification of Inclusion Bodies



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Prepare t e me ia as per t e it instructions

Dissolve the contents of each of the media packets in 1 liter of deionizedwater.

Add 4 ml of glycerol to the Turbo Broth and Power Broth solutions.

Dispense at desired volume into appropriate boles or flasks.

Autoclave at 121C for 15-20 min, depending on the volume per container,and allow to cool.

Dissolve the contents of the Glucose-Nutrient Mix in 100 ml deionizedwater.

Filter sterilize the Glucose-Nutrient Mix using a 0.2 mm filter.

A 50 m o t e steri e G ucose-Nutrient Mix to 1 iter o Hyper Brot

and 20 ml to 1 liter of Glucose M9Y using aseptic technique.

A steri e anti iotics as nee e .

Perform the media screen as follows:

Materials

0 m o eac o t e six i erent cu ture me ia in 250 m a eboomed flasks.

1.

2.

Supplemental Protocol 2: Methods for Increasing Soluble Protein Accumulation

Before embarking on experiments to define a protein refolding regime, it is advisableto first determine whether or not the target protein can be recovered in a solubleand therefore presumably native state. Two relatively simple and quick tests arerecommended; a media screen and induction of chaperone proteins. It is known thatme ium composition can e ect t e accumu ation o recom inant proteins. Li ewise,the relative fraction of soluble protein of an otherwise insoluble product is affectedby an as yet undetermined mechanism. To determine whether medium compositiona ects a particu ar protein, severa me ia ormu ations s ou e screene oraccumulation of the target protein in soluble extracts.

T e o owing protoco is or use wit At enas Me ia Optimization Kit or APFMedia Optimization Kit. The method can be adopted for use with any set of mediaformulations desired.

1.1.

1.2.

1.3.

1.4.

1.5.

1.6.

1.7.

1.8.

2.1.

2.1.1.


Methods for Increasing Soluble Protein Accumulation

14

2.5. Ana yze y SDS-PAGE or purity an ractionate y size exc usionchromatography as needed.


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Was Bu er: 50 mM so ium p osp ate pH 7.5, 150 mM NaC

Lysis Buffer: 50 mM Tris-Cl, 0.2 M NaCl, 2 mM EDTA, proteasenhibitors as needed

Enzyme Stoc So ution: 10 mg/m ysozyme, 1.0 mg/m DNaseI inLysis Buffer

Urea Bu er: 8 M urea, 100 mM Na2HPO4, 10 mM Tris-C pH 7.5 (ors determined for solubilization of the target protein)

2x SDS-PAGE Loa ing Dye: 125 mM Tris-C pH 6.8, 4% SDS(w/v), 0.005% bromphenol blue (w/v), 20% glycerol (v/v), 5% -

ercaptoethanol (v/v)

ris-Glycine SDS-polyacrylamide gel of appropriate composition

Met o s

Inoculate a single colony of the recombinant strain into 10 ml of LBBrot in a s a e as wit a e ooms. Incu ate at 37C overnig t.

Inoculate 50 ml of each of the six media with 5 ml of the overnightu ture. Incu ate t e cu tures at 37C unti t e OD600 reac es 0.6-0.8.

Remove a 15 ml sample (pre-induction), harvest the cells, wash onceith Wash Buffer and collect in a pre-weighed centrifuge tube, and

rocess as in step 2.2.7.

Add inducer and continue incubating for 3 hours.

Remove a 15 m samp e (post-in uction), arvest t e ce s in a pre-eighed centrifuge tube, wash once with Wash Buffer, determine the

ell pellet mass and process as in step 2.2.7.

Harvest the remainder of the culture, wash with 10 ml of wash buffer,etermine the mass of the cell pellet, and store the cell pellets at 80C.

Analyze for expression of the target protein as follows:

Prepare ce extracts as o ows:

Suspend the cell pellets from the pre- and post-in uction samp es in 2 m o Lysis Bu er per gram ocells.

Add lysozyme and DNaseI to 1.0 and 0.1 mg/ml,respectively.

2.1.2.

2.1.3.

2.1.4..

2.1.5.

2.1.6.

2.1.7.

2.2.

2.2.1.

2.2.2.

2.2.3.

2.2.4..

2.2.5.

2.2.6.

2.2.7.

2.2.7.1.

2.2.7.1.1

2.2.7.1.2



15


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Incu ate on ice or 60 min.

Remove a 100 ml sample and reserve. Label wholecell extract.

Lyse t e remaining ce s wit t ree cyc es o reezing(dry ice-ethanol bath, 5 min.) and thawing (37C, 5min).

Clarify the extract by centrifuging at 30,000 x g for 30min at 4C.

Reserve the supernatant, soluble fraction, andsuspend the pellet, insoluble fraction, in 0.5 ml UreaBu er or ot er so u i ization u er.

Determine the protein concentration in each of theractions.

Determine the presence of the target protein in the solubleractions y one o t e o owing means:

Functional Assay Perform a functional assay usingequal amounts of protein in the assay.

Immuno ot or Microp ate Assay Loa equaprotein per lane of a gel, well of a slot/dot blot ormicroplate well. Detect the target protein using aprimary anti o y to an a nity tag or to t e targetprotein.

SDS-PAGE Loa equa amounts o protein perlane. Stain the gel with Coomassie Blue, colloidalCoomassie Blue or silver stain.

Interpretation

Compare t e eve o target protein o taine rom ce s grown

n each of the six media. Select the medium which produces theighest level of soluble target protein per ml of culture.

2.2.7.1.3.

2.2.7.1.4

2.2.7.1.5.

2.2.7.1.6.

2.2.7.1.7.

2.2.7.1.8.

2.2.7.2.

2.3.

2.2.7.2.1.

2.2.7.2.2

2.2.7.2.3.

2.3.1.



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Supplemental Protocol 3: Increased Soluble Protein Accumulation Using Chaperone Induction

Ot er actors w ic can increase t e accumu ation o so u e protein are c aperoninproteins. Chaperones are a class of proteins found in all organisms which play a role

in folding of protein or the refolding of mis-folded proteins. Several studies haveshown that the co-expression of selected chaperones increases the accumulation ofsoluble protein during hyper-expression.1 However, in vitro studies have found thatnot a proteins are acte on y c aperones uni orm y.12 In ot er wor s, w i e t eaccumulation of soluble forms of some proteins can be increased by chaperones, otherproteins are unaffected. At the present time no classification scheme is available toa ow one to pre ict w ic proteins are i e y to e acte on y a give c aperone orset of chaperonins. Therefore, trial and error testing would be needed to identify asuitable chaperone(s) for a given protein. Complicating this is that not all chaperoneproteins are avai a e in su cient quantities or re o ing wor .

As an alternative, Athenas scientists have developed a medium additive,Augme ium, w ic in uces t e expression o c aperones. Rat er t an co-expressing one specific chaperone, Augmedium, causes a sublethal chemical andoxi ative stress w ic resu ts in t e expression o a range o c aperone proteins. Inthis way, prior knowledge of which family of chaperones that act on the target proteinis not needed.

Inoculate 10 ml Turbo Broth, Turbo Prime Broth (or other medium knowno give good yields of the target protein) supplemented with the appropriatenti iotics, wit a sing e co ony o t e expression strain an incu ate overnig t at

37C.

Use t e overnig t cu ture to inocu ate six 250 m a e oom as s e wit0 ml medium each. Incubate at 30C until the density reaches an OD600 of 0.9.

A 0.5, 0.25, 0.125, 0.0625, an 0.03125 m 50x Augme ium to eac o veasks. The sixth flask is the untreated control. Incubate 20 min.

A IPTG (or ot er in ucer as per t e expression system) to 1 mM an incu ateor 3 hours.

Harvest t e cu tures y centri ugation at 3,000 x g or 20 min Store t e pe ets at20C or -80C until processing.

Prepare ce - ree extract y mec anica , c emica or enzymatic isruption. C ari yhe extract by centrifuging at 30,000 x g for 30 min. Reserve the supernatant.

Determine the amount of soluble protein in the supernatant by one of theollowing means:

1.

2.

3.

4.

5.

6.

7.


Increased Soluble Protein Accumulation Using Chaperone Induction

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Functiona Assay Per orm a unctiona assay using equa amounts oprotein in the assay.

Immunoblot or Microplate Assay Load equal protein per lane of a gel,

well of a slot/dot blot, or microplate well. Detect the target protein with aprimary anti o y to an a nity tag or to t e target protein.

SDS-PAGE with Coomassie or silver stain Load equal amounts of proteinin eac ane. Compare t e re ative eve o target protein accumu ate .

Select the level of Augmedium which yields the highest level of target protein.Furt er in ormation is avai a e in t e Augme ium Data S eet.13

7.1.

7.2.

7.3.

8.


Increased Soluble Protein Accumulation Using Chaperone Induction

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References

Baneyx, F. 1999. In vivo folding of recombinant proteins in scherichia coli. In Manual ofIndustrial Microbiology and Biotechnology, 2nd Edition, Demain, A. L. and Davies, J. E., eds.,

ress, as ington, .

n nsen, . . . rincip es t at govern t e o ing o protein c ains. cience. : - .

Rudolph, R. and Lilie, H. 1996. In vitro folding of inclusion body proteins. FASEB 10:49-56.

Clark, E. 1998. Refolding of recombinant proteins. Current Opinion in Biotech. 9:157-163.

Lilie, H. Schwarz, E., and Rudoolph, R. Advances in refolding of proteins produced in . coli.Current Opinion in Biotech. 9:497-501.

Xie, Y., and Wetlaufer, D. B. 1996. Control of aggregation in protein folding: the temperature-eap tactic. rotein ci. : - .

Meada, Y., Uedaa, T. and Imoto, T. 1996. Effective renaturation of denatured and reducedimmunog o u in in vitro wit out assistance o c aperone. rotein ng. : - .

oo ing, . . an reiser, . . . ig -per ormance size-exc usion c romatograp y oproteins. n ig - er ormance iqui romatograp y o epti es an roteins: eparation,Analysis and Conformation. Mant., C. T. and Hodges, R. S. eds., CRC Press, Boca Raton, FL.

nge ar t, . . ize-exc usion c romatograp y o proteins. i .

ontgomery. . esign an ana ysis o xperiments, t ition, o n i ey an ons,New York.

arston, . . ., an art ey, . . . o u i ization o protein aggregates. et o s inEnzymology 182:264-277.

Horwich, A. L., Weber-Ban, E. U., and Finley, D. 1999. Chaperone rings in protein refoldingand degradation. Proc. Natl. Acad. Sci. USA. 96:11033-11040.

Augmedium Data Sheet, www.athenaes.com.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

References

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Technical Assistance

T e scienti c sta o t e At ena Enzyme Systems are specia ists in t e expressionf recombinant proteins in microbial systems. They have extensive expertise in

aspects o protein expression rom t e construction o expression vectors to t eommercial production of recombinant proteins. No maer what your question,please feel free to ask them for help. A technical support scientist can be reached atupport@at enaes.com.

Product Use Limitations

T e Quic Fo Protein Re o ing Kit was esigne an is so or researc usenly. It should not be used for human diagnosis or drug use or administered to

humans unless expressly cleared for that purpose by the appropriate regulatoryuthorities in the country of use. All due care and aention should be exercised in the

handling of the materials contained in the kit.

Product Warranty

At enaES guarantees t e qua ity an per ormance o t e me ia an reagentscontaine in t is it or t e cu tivation o E. co i. T e suita i ity o a me iumormu ation or a itive or a particu ar use is t e responsi i ity o t e en user. No

guarantee is made that a given protein will be expressed when applying this kit.AthenaES will replace the product free of charge if it does not conform to the statedspecifications. Notice for replacement must be received within 60 days of opening theproduct.

Technical Assistance

Product Use Limitations

Product Warranty

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Ordering Information

To place an order:

Phone: 1-888-892-8408Fax: 410-455-1155

Or visit our website to order through one of our international distributors.

When placing an order, please provide the following:

- Institution name and customer service account

- Purchase order number

- Catalog number(s) or names of products and quantity of item(s)

- Billing and shipping address

- Contact name and telephone number

Delivery:

Telephone orders received Monday through Friday before 12 noon will be shipped that

day. All other orders will be shipped the next business day, unless otherwise stipulated.

Email: [email protected]: www.athenaes.com

Cat. No. Size Cat. No. Size Product

0612-1 00 mL 613-1 1L uickFold Protein Refolding Buffer 1

0612-2 00 mL 613-2 1L uic Fo Protein Re o ing Bu er 2





- 00 mL - 1L uic Fo Protein Re o ing Bu er 7



- 00 mL - 1L uic Fo Protein Re o ing Bu er 10






QuickFold Individual Protein Refolding Buffers

Ordering Information

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