Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
RECOMBINANT PROTEINS
Beáta ScholtzMolecular Therapies- Lecture 3
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
1.1 OVERVIEW: PROTEIN PHARMACEUTICALS
1.2 CELL-FREE SYSTEMS: IN VITRO TRANSCRIPTION AND TRANSLATION
1.3 EXPRESSION OF RECOMBINANT PROTEINS IN CELL CULTURE
1.4 NON-PROKARYOTIC EXPRESSION SYSTEMS1.4.1 Pichia pastoris1.4.2 Protein expression in insect cells1.4.3 Mammalian expression systems
1.5 PURIFICATION OF RECOMBINANT PROTEINS
RECOMBINANT PROTEINS
The aim of this lecture is to describe the in vitro and in vivo systems utilized forexpression of recombinant proteins, and discuss the advantages and disadvantages of these systems. We will also discuss the basics of affinity-tag based protein purification.
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Pure protein preparations
Uses: medicine and researchSources: • natural protein mixtures - human/animal/fungi/plant• artificial preparations - synthetic peptides, recombinant proteins
Insulin Pigs or cattle (pancreas)Factor VIII Human blood (donated)Human growth hormone Human brainsCalcitonin SalmonAnti-venom Horse or goat blood
Protein pharmaceutical Natural Source
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Equipment used for blood fractionation
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B. Rogge Box jellyfish, Australia
Lonomia caterpillar, BrasilR. Morante
Black scorpion, Arabia
P-A. Olsson
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Protein pharmaceuticals
Natural sources are often rare and expensive Difficult to keep up with demandHard to isolate productMay lead to immune reactions (diff. species)Viral & pathogen contamination
Most protein pharmaceuticals today are produced recombinantlyCheaper, safer, abundant supply
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Peptide drugs
Many hormones are actually small peptides (2-40 amino acids)Calcitonin (32 residues)
Thyroid hormone to enhance bone massOxytocin (9 residues)
Pituitary hormone to stimulate laborVasopressin (9 residues)
Pituitary hormone for antidiuretic/vasoconstriction
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Peptide drugs
Small enough to synthesize using solid phase chemistry (SPPS)Method developed by Bruce Merrifield in 1960’s (won Nobel
prize)Very efficient synthesis (>99%/couple)Still: 50 residue peptide, 99% coupling
Yield = 0.9950 = 60.5%Technique limited to small peptides
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Recombinant proteins
Developed in 1970’s &1980’sPaul Berg (1973) restriction enzymesHerbert Boyer (1978) cloning human insulin into E. coli –
GenentechFour general approaches
Expression in cell-free systems Expression in isolated cells Expression in transgenic plants/animals Gene therapy in humans
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Cell-free systems:In vitro transcription and translation
• Rapid identification of gene products• Functional analyses• Analyze protein-protein interactions• Study protein folding• Incorporate modified amino acids for functional studies• Engineer truncated gene products
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Advantages over in vivo gene expression:When the protein is:
toxic to the host cellinsoluble or forms inclusion bodies
degraded rapidly by intracellular proteasesSpeed and directness of all proceduresAbsence of constraints from a living cellPure product Disadvantages over in vivo gene expression:Lack of cellular membranesLack of post translational modifications
Cell-free systems:In vitro transcription and translation
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Components for in vitro transcription
• Linearized DNA template• Phage RNA polymerase• 4dNTP• Buffer
1998 by Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walter. Published by Garland Publishing.
In vivo In vitro
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Phage RNA polymerases
Phage Polymerase Host of Phage Promoter Sequence
T7 RNA polymerase E. coli 5’TAATACGACTCACTATAGGG 3’
T3 RNA polymerase E. coli 5’AAATTAACCCTCACTAAAGGG3’
SP6 RNA polymerase Salmonella typhimurium 5’AATTTAGGTGACACTATAGAA3’
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Characteristics of RNA polymerases
RNA polymerases proceed at a much slower rate than DNA polymerases.
RNA pol (50-100 bases/sec)
DNA pol (1000 bases/sec)
The fidelity of RNA synthesis is much lower than that of DNA.
RNA polymerases do not contain proofreading mechanisms.
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DNA template
Plasmids Many commonly used cloning vectors contain phage polymerase promoters outside of the multiple cloning site.
PCR ProductsPrimer must contain promoter
Oligonucleotides
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Linearization of template
• Plasmids: no RNA polymerase termination signal; templates are linearized
• PCR template: termination signal in the amplified region OR in the primer
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Translation in eukaryotic cells
1998 by Alberts et al.
Published by Garland Publishing.
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• tRNA & aminoacyl-tRNA synthetases• Ribosomes• Amino acids• ATP, GTP• Initiation, elongation, and termination factors• Buffer• RNA template
Components for in vitro translation
Much more complex than transcriptionCannot be mixed from a few isolated components
Always provided as crude extract of cells
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Common in vitro translation systems
Rabbit reticulocyte lysate
Wheat germ extract
E. coli extract
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Rabbit reticulocyte lysates
Reticulocytes: immature red blood cellsno nuclei (DNA)complete translation machinery, for extensive globin synthesis
Endogenous globin mRNA can be eliminated by incubation with a Ca2+dependent micrococcal nuclease. The nuclease is later inactivated by EGTA.
Low background
Efficient utilization of exogenous RNAs, even at low concentrations
Low nuclease activity
Capable of synthesizing large amounts of full-length products
Capable of translating either capped or uncapped RNAs
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Wheat germ lysates
Low background incorporation due to low levels of endogenous mRNA
Recommended for translation of RNA containing small fragments of double-stranded RNA or oxidized thiols, which are inhibitory to the rabbit reticulocyte lysate
Generally more cap dependent than reticulocyte systems
Often preferable when synthesizing relatively small proteins (12-15kDa) that comigrate with globin, which is abundant in reticulocyte extracts
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E. coli lysates
Simple translational apparatus and less complicated initiation control mechanisms
BUT: bacterial extracts contain nucleases that rapidly degrade most exogenous RNAs
Extract must be incubated during preparation so that excess endogenous mRNA is translated and subsequently degraded
The exogenous product is easily identifiable
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Translation systems
Two approaches to cell free protein synthesis: Standard translation systems (reticulocyte and wheat extracts) use RNA as a
template
Linked or coupled transcription+translation systems start with DNA templates
Important elements for translation:= Eukaryotic translation signal: 5’-GCCACCAUGG-3’ “Kozak”
sequence, if eukaryotic cell free translation system is used= Prokaryotic translation signals: 5’-UAAGGAGGUGA-3’
Shine- Delgarno (SD) , if prokaryotic cell free translation system is used
Linked system: tube 1.=transcription, tube 2.= translation.= Each can be optimized separately.
Coupled system: both reactions in the same tube
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Main steps of recombinant protein production
Identification/Isolation of gene of interestCloning of gene into plasmid
Plasmid: expression vectorTransformation into host cellsGrowth of cells through fermentation
Plasmid: source of DNA templatefor transcriptionIn vitro transcriptionIn vitro translation
Isolation & purification of protein
In vivo Cell free
Formulation of protein product Research
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Escherichia coli/ Other bacteriaPichia pastoris/ Other yeastInsect cell culture (Baculovirus)Mammalian cell culturePlants
Sheep/cows/humans(transgenics and gene therapy)
Recombinant protein expression in cells or organismsTÁMOP-4.1.2-08/1/A-2009-011
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Expression system selection
Choice depends on size and character of protein
Large proteins (>100 kD)? Choose eukaryote Small proteins (<30 kD)? Choose prokaryote High yields, low cost? Choose E. coli Post-translational modifications essential? Choose yeast,
baculovirus or other eukaryote Glycosylation essential? Choose baculovirus or mammalian
cell culture
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Characteristics of (plasmid) vectors
1. Must be compatible with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells)
2. Features :• strong promoter/inducible promoter• transcription START sequences• ribosome binding sites• termination sequences, polyA signal sequence• affinity tag or solubilization sequences
• multi-enzyme restriction site• origin of replication (ORI) • bacterial selectable marker (Amp or Tet) • eukaryotic selectable marker• recombination sequences
proteinexpression
cloning, plasmidpropagation
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Promoter selection
• Constitutive - everywhere, all the time
• Tissue- or developmental stage-specific - selected cell types, specific timing
• Inducible - specific timing, can avoid toxicity to host
• Synthetic
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Inducible promoters: Tet-off systemTÁMOP-4.1.2-08/1/A-2009-011
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(faster response)
Inducible promoters: Tet-on system
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Steroid hormone induction: adenovirus promoterglucocorticoid response elementinducer: dexamethasone
Tetracycline operon: CMV promoterTet operator sequence, Tet repressor proteininducer/repressor: tetracycline
Ecdyson-inducible system: requires two vectorsSV40 promoterhuman RXR receptor and Drosophilaecdyson receptor (VgEcR) = transcriptionfactor heterodimerActivator of transcription factor: pronasteroneANice dose response
Synthetic promoters, inducible systemsTÁMOP-4.1.2-08/1/A-2009-011
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Bacterial expression systems
Grows quickly (8 hrs to produce protein)High yields (50-500 mg/L)Low cost of media (simple media constituents)Low fermentor costs
Difficulty expressing large proteins (>50 kD)No glycosylation or signal peptide removalEukaryotic proteins are sometimes toxicCan’t handle S-S rich proteins
Advantages Disadvantages
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Promoter selection for prokaryotes
Promoter type Expression level Regulator/inducer Main characteristicslac promoter low/middle IPTG Low level intracellular expression
trc and tac promoter moderatly high IPTG Higher expressionT7 RNA polymerase promoter very high IPTG Basal level depends on strain
T7-lac system for tight controlHigh level induction
TetA promoter/operon middle/high tetracycline Low basal levelTight regulation
Independent of metabolic statePhage promoter pL moderatly high temperature shift Very low basal level
Temperature sensitive host neededPPBAD promoter low/high L-arabinose Very low basal level
Tight regulationFine-tuning, dose dependent
rhaPBAD promoter low/high L-rhamnose Very low basal levelTight regulation
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Cloning & transforming in yeast cells
Pichia pastorisSaccharomyces cerevisiae
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Pichia pastoris
Yeast are single celled eukaryotesBehave like bacteria, but have key advantages of eukaryotesP. pastoris is a methylotrophic yeast that can use methanol as its
sole carbon source (using alcohol oxidase)Has a very strong promoter for the alcohol oxidase (AOX) gene
(~30% of protein produced when induced)
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Pichia expression system
Grow quickly (8 hrs to produce protein)Very high yields (50-5000 mg/L)Low cost of media (simple media constituents)Low fermentor costs
Can express large proteins (>50 kD)Glycosylation & signal peptide removalHas chaperonins to help fold “tough” prtnsCan handle S-S rich proteins
Advantages More advantages
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Pichia pastoris cloning
Uses a special plasmid that works both in E. coli and yeastOnce gene of interest is inserted into this plasmid, it must be
linearizedTransfect yeast cells with linear plasmidDouble cross-over recombination event occurs to cause the gene
of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be
Now gene of interest is under control of the powerful AOX promoter
Stable transfectant
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Cloning a gene into Pichia vector TÁMOP-4.1.2-08/1/A-2009-011
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Baculovirus/insect cell expression systems
Bastiaan (Bart) Drees
Spodoptera f. larvaSpodoptera frugiperda
Sf9 cells and baculovirus
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Baculovirus life cycle
1.
2.
3a.
3b.
4a.
4b.
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Baculovirus life phases in culture
1. Early phase: cell entry, shutting down host gene expression viral protein synthesis
2. Late phase: viral DNS replication, virus assembly, release of viral particles from cell (peak:18-36 hrs post-infection) Also used to prepare viral stock
3. Very late phase: polyhedrin and p10 genes are expressed, viruses embedded in polyhedrin form occlusion bodies. Cell lysis. (24-96 hrs post-infection) Used for protein production
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Baculovirus mediated protein expression in insect cells
Autographica californica multiple nuclear polyhedrosis virus (Baculovirus)
Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
Uses super-strong promoter from the polyhedrin coat protein to enhance expression of proteins while virus resides inside the insect cell - protein is not required for infection or viral life cycle
Secreted proteins better expressed by stably transfected insect cell lines, from the ie-1 promoter(infection interferes with secretory pathways)
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Baculovirus expression system workflow
1. Cloning gene of interest into baculovirus genome
2. Use recombinant baculoviral DNA to transfect insect cells
3. Collect viral particles from insect cell culture supernatant
4. Test viral stock titer, freeze stocks
5. Infect new insect cell culture
6. Harvest cells (with occlusion bodies)
Note: not a stable cell line!
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Cloning a gene into baculovirus (AcMNPV) vector
5’ 3’
Transfer vector
x x
Cloned gene
modified AcMNPV DNA,“Bacmid” maintained in E. coli
5’ 3’Cloned gene
RecombinantAcMNPV bacmid
Site-specific transposition
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Gene of Interest
Tn7R polyhedrin promoter Gent+ Tn7L
Transfer vector with insert
Gene of Interest
Tn7 R
PpH Tn7 L
Bacmid with insert
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128bp 145bpMini att Tn7M 13 forward M 13 reverse
Tn7R GOI Tn7L
Bacmid DNA
Transposition into bacmid
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Baculovirus expression system
Grow very slowly (10-12 days for set-up)Cell culture is only sustainable for 4-5 daysSet-up is time consuming, not as simple as yeast
Can express large proteins (>50 kD)(Mostly) Correct glycosylation & signal peptide removalHas chaperonins to help protein foldingVery high yields, cheap
Disadvantages Advantages
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Baculovirus successes
Alpha and beta interferonAdenosine deaminaseErythropoietinInterleukin 2Poliovirus proteinsTissue plasminogen activator (TPA)
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Mammalian expression systemsTÁMOP-4.1.2-08/1/A-2009-011
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Mammalian expression systems
Selection takes time (weeks for set-up)Cell culture is only sustainable for limited period of timeSet-up is very time consuming, costly, modest yields
Can express large proteins (>50 kD)Correct glycosylation & signal peptide removal, generates authentic proteinsHas chaperonins to help protein folding
Disadvantages Advantages
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Mammalian expression system
Gene initially cloned into plasmid, and propagated in bacterial cells
Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
Mammalian cells transformed by electroporation (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes
Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression & integration of DHFR and the gene of interest
Stably transfected cell lines are generated - long term culturing
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Characteristics of mammalian expression vectors
Recombinant gene expression requires multiple elements in the vector:
• promoter (general or tissue-specific)• enhancer• polyA signal• intron - may enhance expression• selection marker (ampicylin, neomycin, DHFR etc.)• Frequently used promoters: simian virus 40 (SV40) (strong promoters) papovavirus
Rous sarcoma virushuman cytomegalovirus (CMV)
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Methotrexate (MTX) selection
Gene of interest DHFR
TransfectDHFR minus cells
Grow innucleosidefree medium
Culture acolony of cells
Grow in0.05 uM Mtx
Culture acolony of cells
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Grow in0.25 uM Mtx
Grow in0.5 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed athigh level inCHO cells
Methotrexate (MTX) selection
Multiple rounds of selection, increasing MTX concentration
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Mammalian cell successes
Factor IXFactor VIIIGamma interferonInterleukin 2Human growth hormoneTissue plasminogen activator (TPA)
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Purification of recombinant proteins
Application Required Purity
Therapeutic use, in vivo studies Extremely high > 99%
Biochemical assays, X-ray crystallography High 95-99%
N-terminal sequencing, antigen for antibody production, NMR Moderately high < 95%
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Purification of recombinant proteins
All proteins are different
Size
Hydro-phobicity Charge
Activity
BEHAVIOUR
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Conventional purification strategy
• Use different properties of protein in purification scheme
• Multiple intermediate steps may be required
• Need to detect low amounts
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Affinity-tag based purification strategy
• Fusion proteins withaffinity tag
• Tag: peptide or protein
• Tag binds something veryselectively and w. highaffinity
• Very effective purificationin initial step
• Tag can be used for detection
• Tag can be cleaved off
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gene for protein of interest insert affinity tag sequence
introduceinto cells
Tagged protein
Purification of tagged protein
Immunolocalization of protein
Other interacting proteins
Affinity-tagging of recombinant proteinsTÁMOP-4.1.2-08/1/A-2009-011
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Solid matrix
Nickel ion (Ni2+)
Poly-histidineon protein
His-Ni2+ stable complex at near-neutral aqueous conditions
Histidine tagTÁMOP-4.1.2-08/1/A-2009-011
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Making proteins bind nickelTÁMOP-4.1.2-08/1/A-2009-011
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His-tag based purification strategyTÁMOP-4.1.2-08/1/A-2009-011
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Examples for affinity and epitope tags
His-tag: N-or C-terminal 6xHistidin, binds to Ni-resin• purification
T7-tag: starting sequence for T7 gene (11 amino acids)• enhancer for translation
S-tag: ribonuclease A S-peptid (15 amino acids)• detection, isolation: biotinylated S-protein, S-protein affinity
Strep-tag: C-terminal AWRHPQFGG sequence (affinity to streptavidin) purification
Epitope-tags: recognised by good antibodies (usually monoclonal)• FLAG-tag (NYKNNNNK)• c-myc-tag (QGKLISGGNL)
TAP-tag: „tandem-affinity purification”, calmodulin-binding protein and protein A both fused to protein of interest
• very good system to study protein-protein interactions
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Fusion proteins in prokaryotic expression systems
Proteins expressed in E. coli are often produced as fusion proteins:
• function of the protein in bacteria is not of interest
• mammalian protein is not expressed effectively by itself
• bacterial fusion partner, (e.g. GST) on the other hand, is expressed effectively – fusion protein is likely to be expressed well, too • one-step purification from bacterial lysate
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Bacterial fusion protein systems
Glutathion-S-transferase: 26 kDa proteinSchistosoma japonica gene productpGEX vector-seriesfast isolation on glutathion-resin
Maltose-binding protein: E. coli malE gene productpMEL vector-seriessolation on maltose affinity column
Thioredoxin 17 kDa protein, heat-stable, very goodsolubilityRibonucleotide-reductase reducing enzymeE. coli trxA gene product pTrxFus vector
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Glutathione-S-transferase fusion protein expression system
pGEXLac inhibitorgene
Ampicyllin resistancegene
Lac promoter
GST
Polylinker orMulticloning site
Ori
Repressorprotein
IPTG
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Which tag to use?
Specificity of binding interaction
Cost of resin
Native vs. denaturing elution
Presence of metals
Expression level, solubility & toxicity of target protein
Tag removal
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Tag removal
NH2– proteintag linker
DDDDK
protease
Linker/cleavagestrategy selection:
• effect on structure• effect on function• flexibility• protein 1° sequence• removal of protease
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Excision Site Cleavage Enzyme Comment
D-D-D-D-KX enterokinase active: pH 4.5-9.5, 4-45°CX cannot be P
secondary cleavage sites
I-D/E-G-RX factor Xa protease X cannot be P/Rsecondary cleavage sites
L-V-P-RG-S thrombin biotynilated form availablesecondary cleavage sites
E-N-L-Y-F-QG TEV protease active: wide range of THis-tagged form available
L-E-V-L-F-QG-P PreScissionTM protease
engineered with GST tag
Tag removalTÁMOP-4.1.2-08/1/A-2009-011
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crude
His-resin I
tag cleavage
His-resin II
gel filtration
Pure protein
Purification protocol : as few steps as possible
• His-resin I usually provides a major step of the purification
• His-resin II removes cleaved-off His-tag and persistent contaminant proteins in E.coli host
• Gel-filtration – “polishing”
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