recombinant protein production
DESCRIPTION
Recombinant Protein Production. Introduction to Expression Systems Core Facility of Recombinant Protein Production, National Research Program for Genomic Medicine. functional studies structural studies vaccine/antigen/antibodies therapeutic drug industrial enzymes for reaction. - PowerPoint PPT PresentationTRANSCRIPT
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Recombinant Protein Production
- Introduction to Expression Systems
- Core Facility of Recombinant Protein Production, National Research Program for Genomic Medicine
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Recombinant Protein Production
• functional studies• structural studies• vaccine/antigen/antibodies • therapeutic drug• industrial enzymes for reaction
-Application
• over-expression to get enough amount• easy purification
-Why?
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Application: Drug Discovery
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Application: therapeutic proteins
• Actimmune (If )• Activase (TPA)• BeneFix (F IX)• Betaseron (If )• Humulin• Novolin• Pegademase (AD)
• Epogen• Regranex (PDGF)• Novoseven (F VIIa)• Intron-A• Neupogen• Pulmozyme• Infergen
•Now more than 200 approved peptide and protein pharmaceuticals on the FDA list (http://www.accessexcellence.org/RC/AB/IWT/The_Biopharmaceuticals.html)
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Application: structural genomics
Bioinformatics
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Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications
Principle in Protein Production
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Protein Expression Bottleneck
Cloning
Expression
Purification
Crystallography
DNA
Enzymology
• Protein Biochemistry– soluble, purifiable protein
• Enzymology– soluble, active protein– 0.1-10 mg of protein
• Crystallography– soluble, crystallizable protein– 5-100 mg of protein
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Bottlenecks to efficient protein expression in E. coli
Promoter choice and design
Inefficient transcription No or little protein synthesized
Codon usageTranscript stabilityTranscript secondary structure
Improper secondary, tertiary or quaternary structure formationInefficient or improper disulfide bridge formationInefficient isomerization of peptidyl-prolyl bonds
Inefficient translation No or little protein synthesized
Inefficient folding (cytoplasmic or periplasmic)
Inefficient membrane insertion/translocation
Toxicity Cell death
Aggregation or degradation
Aggregation or degradation
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Protein Expression and Purification
• Isolation of gene of interest• Introduction of gene to expression vector• Transformation into host cells• Growth of cells through fermentation• Isolation & purification of protein
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Cloning and expression of target gene:
+
Recombinant Vector
Gene of Interest
Expression Vector
Expression of Fusion Protein
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Cloning Process
• Gene of interest is cut out with restriction enzymes (RE)
• Host plasmid (circular chromosome) is cut with same REs
• Gene is inserted into plasmid and ligated with ligase
• New (engineered) plasmid inserted into bacterium (transform)
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Cloning (Details)
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Cloning (Details)
protein
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Recombinant Protein Expression Systems
• Escherichia coli• Other bacteria• Pichia pastoris• Other yeast• Baculovirus• Animal cell culture• Plants• Sheep/cows/humans• Cell free
Polyhedra
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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– Glycosylation essential? Choose baculovirus or mammalia
n cell culture– High yields, low cost? Choose E. coli– Post-translational modifications essential? Choose yeast,
baculovirus or other eukaryote
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Which Vector?
• Must be compatible with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells)
• Needs a good combination of– strong promoters– ribosome binding sites– termination sequences– affinity tag or solubilization sequences– multi-enzyme restriction site
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Plasmids and Vectors
• Circular pieces of DNA ranging in size from 1000 to 10,000 bases
• Able to independently replicate and typically code for 1-10 genes
• Often derived from bacterial “mini” chromosomes (used in bacterial sex)
• May exist as single copies or dozens of copies (often used to transfer antibiotic resistance)
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Key Parts to a Vector
• Origin of replication (ORI) – DNA sequence for DNA polymerase to replicate the plasmid
• Selectable marker (Amp or Tet) – a gene, when expressed on plasmid will allow host cells to survive
• Inducible promoter – Short DNA sequence which enhances expression of adjacent gene
• Multi-cloning site (MCS) – Short DNA sequence that contains many restriction enzyme sites
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A Generic Vector
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Which Vector?
• Promoters– arabinose systems (pBAD), phage T7 (pET), Trc/Tac promot
ers, phage lambda PL or PR• Tags
– His6 for metal affinity chromatography (Ni)
– FLAG epitope tage DYKDDDDK– CBP-calmodulin binding peptide (26 residues)
– E-coil/K-coil tags (poly E35 or poly K35)
– c-myc epitope tag EQKLISEEDL– Glutathione-S-transferase (GST) tags– Celluluose binding domain (CBD) tags
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Gene Introduction (Bacteria)
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Bacterial Transformation
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Bacterial Transformation
• Moves the plasmid into bacterial host• Essential to making the gene “actively” express the protei
n inside the cell• 2 routes of transformation
– CaCl2 + cold shock
– Electroporation• Typical transformation rate is 1 in 10,000 cells (not very ef
ficient) for CaCl2, but 1 in 100 for electroporation
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Electroporator
25 microfarads = 2500 V@ 200 ohms for 5 ms
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Electroporation
• Seems to cause disruption in cell membrane
• Reconstitution of membrane leads to large pores which allow DNA molecules to enter
• Works for bacteria, yeast and animal cells
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Bacterial Systems
• Grow 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 removal
• Eukaryotic proteins are sometimes toxic
• Can’t handle S-S rich proteins
Advantages Disadvantages
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Cloning & Transforming in Yeast Cells
Pichia pastoris
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Pichia Pastoris
• Yeast are single celled eukaryotes• Behave like bacteria, but have key advantages of euka
ryotes• P. pastoris is a methylotrophic yeast that can use meth
anol as its sole carbon source (using alcohol oxidase)• Has a very strong promoter for the alcohol oxidase (AO
X) gene (~30% of protein produced when induced)
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Pichia Cloning
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Pichia Pastoris Cloning• Uses a special plasmid that works both in E. coli and Yeast• Once gene of interest is inserted into this plasmid, it must be lineari
zed (cut open so it isn’t circular)• Double cross-over recombination event occurs to cause the gene o
f 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
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Pichia Systems
• 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 removal
• Has chaperonins to help fold “tough” prtns
• Can handle S-S rich proteins
Advantages More advantages
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Baculovirus Expression
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Baculovirus Expression
• Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
• Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)
• Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell
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Baculovirus Expression
~12 days
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Baculovirus (AcMNPV) Cloning Process
5’ 3’
Transfer vector
Polyhedrin gene
x x
Cloned gene
AcMNPV DNA
5’ 3’Cloned gene
RecombinantAcMNPV DNA
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Baculovirus Systems
• Grow very slowly (10-12 days for set-up)
• Cell culture is only sustainable for 4-5 days
• Set-up is time consuming, not as simple as yeast
• Can express large proteins (>50 kD)
• Correct glycosylation & signal peptide removal
• Has chaperonins to help fold “tough” prtns
• Very high yields, cheap
Disadvantages Advantages
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Mammalian Expression Systems
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Mammalian Cell-line Expression
• Sometimes required for difficult-to-express proteins or for “complete authenticity” (matching glycosylation and sequence)
• Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line
• Vectors usually use SV-40 virus, CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene
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Mammalian Expression
• Gene initially cloned and plasmid propagated in bacterial cells
• 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
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Methotrexate (MTX) Selection
Gene of interest DHFR
Transfectdfhr- cells
Grow inNucleosideFree medium
Culture aColony of cells
Grow in0.05 uM Mtx
Culture aColony of cells
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Methotrexate (MTX) Selection
Grow in5.0 uM Mtx
Grow in0.25 uM Mtx
Culture aColony of cells
Culture aColony of cells
Foreign geneexpressed inhigh level inCHO cells
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Mammalian Systems
• Selection takes time (weeks for set-up)
• Cell culture is only sustainable for limited period of time
• Set-up is very time consuming, costly, modest yields
• Can express large proteins (>50 kD)
• Correct glycosylation & signal peptide removal, generates authentic proteins
• Has chaperonins to help fold “tough” prtns
Disadvantages Advantages
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Conclusion
• Isolation of gene of interest• Introduction of gene to expression vector• Transformation into host cells• Growth of cells through fermentation• Isolation & purification of protein
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National Research Program for Genomic Medicine
Core Facility of Recombinant Protein Production重組蛋白質生產核心設施 D1
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Expression systems
• E. coli• Baculovirus• Yeast• Cell-free • Mammalian cell
( not open for service)
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SYSTEMS Advantages Disadvantages
E. coli
•Parallel cloning•Fast•Ease of use•Low cost
•Poor expression•Low solubility •Lacking post-translational modifications
Cell-free •Faster •Skips cell transformation, growing, and lysis
•Low protein yield•Expensive•Tricky to optimize the lysate •and expression conditions
Yeast •Glycosylation•Efficient Economical•Protein with disulfide bonds
•Different glycosylation to mammalian cells
Baculovirus•Most proper eukaryotic •Duration of expression limited to infection period
•Virus production contains numerous steps•Maintain high virus titers
Mammalian cells•Native environment for mammalian proteins
•Lower protein yield•Expensive
Expresssion Systems
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E. coli - the most popular expression system
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• growth condition (e.g. temperature)• codon usage• host strain• fusion to carrier protein
-improvement
• poorly expressed• protein insoluble- inclusion bodies• expressed and soluble: 20-30%
-challenge
E. coli Expression System
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Rationale
1. Increase the expression level and solubility of
target protein with protein tags.
2. Simultaneously, parallel screening different
fusion tags.
3. Has potential for automating gene cloning.
Publication Protein Science (2002), Shih YP et. al.,
11:1714-1719.
E. Coli Expression System
parallel screening for soluble proteins
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Sticky-end PCR
E. coli
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Parallel Gene Cloning
E. coli
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Parallel screening for soluble protein
E. coli
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Statistical analysis of soluble protein ratio
E. coli
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E. coli Expression System - Modified version
To improve consistency and convenience, we now modify the above vectors to include a hexa-His tag and a Factor Xa cleavage site at the N-terminus of each protein expressed in E. coli
EcoR I Xho IPromotor
Fusion tag ThrombinHis*6 FXaTarget
ProteinHis*6
Terminator
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技術比較說明融合蛋白質的選擇類似,主要是 cloning的差別
Donor vector
我們使用 Sticky-end PCR 的方法,不必經過 Sub-cloning 即可 parallel cloning
PCR Denature
Re-nature
PCR Ligation Co-transformationPurify plasmid
他人已使用商品化的策略; Gateway Technology (Invitrogen)
E. coli
Hammarström et al. Protein Science (2002), 11:313–321
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• The method introduces sticky-end to target genes, without using restriction enzymes.
• Well-induced and highly soluble recombinant proteins : 80% success
E. coli
E. Coli Expression System Summary
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Alternative Expression Systems
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Baculovirus expression system
Bright filed UV merged
- EGFP expressed in baculovirus transfected insect cell
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1: Negative control2: Positive control (GFP)3: Hpps component II4: Hyaluronan synthase5: Rubber prenyl transferase6: Marker
1 2 3 4 5 6
Cell-free expression system Yeast expression system
1: Marker2: N3D TPL-2 using horseshoe crab signal peptide3: N3D TPL-2 using pichia signal peptide
1 2 3
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服務項目介紹
http://proteome.sinica.edu.tw/prod_services_01.asp
服務編號 服務名稱 規格
收費 (台幣)
D1-1水溶性重組蛋白質之表達篩選 (大腸桿菌系統 )
Transformed E coli. strain
14,000
D1-2水溶性重組蛋白質之表達篩選 (大腸桿菌系統 )技術轉移 依需求訂定
D1-3 酵母菌系統之重組蛋白表達篩選 Pichia system 27,500
D1-4無細胞之重組蛋白表達篩選(使用本系統專用載體 )
Cell free system 18,500
D1-5無細胞之重組蛋白表達篩選(自備質體 )
Cell free system(自備質體 )
7,300
D1-6 桿狀病毒系統之重組蛋白表達篩選 Baculovirus expression system
36,300
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SYSTEMS Advantages Disadvantages
E. coli (14,000 NT$)
•Parallel cloning•Fast•Ease of use•Low cost
•Poor expression•Low solubility •Lacking post-translational modifications
Cell-free (18,500/7,300 NT$)
•Faster •Skips cell transformation, growing, and lysis
•Low protein yield•Expensive•Tricky to optimize the lysate •and expression conditions
Yeast (27,500 NT$)
•Glycosylation•Efficient Economical•Protein with disulfide bonds
•Different glycosylation to mammalian cells
Baculovirus(36,300 NT$)
•Most proper eukaryotic •Duration of expression limited to infection period
•Virus production contains numerous steps•Maintain high virus titers
Mammalian cells•Native environment for mammalian proteins
•Lower protein yield•Expensive
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Expression test in E. coli
Parallel Cloning
Soluble
Insoluble / posttranslational modification required
Protein Purification
Yeast system Baculovirus system
in vitro expression systems
standard
additional charge
Service Requested
Protease cleavage to remove tag
Flow chart of protein production
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Self-cleavage of fusion protein in vivo using TEV protease to yield native protein
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• TEVP intracellular processing system
-our approach
• fusion carriers cannot be processed by proteolysis• cleaved products aggregate immediately • cleaved products contain extraneous a.a. residues
-challenge to fusion protein method separation of passenger target protein from the fusion carrier
tobacco etch virus protease (TEVP)
-Glu(P6)-P5-P4-Tyr(P3)-P2-Gln(P1)- -P1'-
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In vivo cleavage of fusion proteins.
TEVP intracellular processing system
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Different amino acid residues at the P1' position
TEVP intracellular processing system
- even with Pro in the P1' position
- more effective than an intermolecular enzymatic reaction
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all six vectors successfully carried out intracellular cleavage
TEVP intracellular processing system
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• introduce cloning sites to target genes, without using restriction enzymes.
• produce native proteins with original amino termini in vivo via intracellular self-cleavage
• skip tedious optimization of cleavage conditions
TEVP intracellular processing system Summary