Recombinant Protein Production

Recombinant Protein Production

- Introduction to Expression Systems

- Core Facility of Recombinant Protein Production, National Research Program for Genomic Medicine

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?

                Application: Drug Discovery

               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)

                Application: structural genomics

Bioinformatics

Bioinformatics

Target identification and cloning

Protein expression test

Protein purification and production

Applications

Principle in Protein Production

                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

               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

               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

Cloning and expression of target gene:

+

Recombinant Vector

Gene of Interest

Expression Vector

Expression of Fusion Protein

                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)

                Cloning (Details)

                Cloning (Details)

protein

                Recombinant Protein Expression Systems

• Escherichia coli• Other bacteria• Pichia pastoris• Other yeast• Baculovirus• Animal cell culture• Plants• Sheep/cows/humans• Cell free

Polyhedra

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

                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

                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)

                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

                A Generic Vector

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

                Gene Introduction (Bacteria)

                Bacterial Transformation

                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

                Electroporator

25 microfarads = 2500 V@ 200 ohms for 5 ms

                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

                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

                Cloning & Transforming in Yeast Cells

Pichia pastoris

                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)

                Pichia Cloning

                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

                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

                Baculovirus Expression

                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

                Baculovirus Expression

~12 days

               Baculovirus (AcMNPV) Cloning Process

5’ 3’

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA

5’ 3’Cloned gene

RecombinantAcMNPV DNA

                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

Mammalian Expression Systems

               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

                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

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

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

                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

                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

National Research Program for Genomic Medicine

Core Facility of Recombinant Protein Production重組蛋白質生產核心設施 D1

                Expression systems

• E. coli• Baculovirus• Yeast• Cell-free • Mammalian cell

( not open for service)

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

E. coli - the most popular expression system

• 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

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

Sticky-end PCR

E. coli

                Parallel Gene Cloning

E. coli

Parallel screening for soluble protein

E. coli

                Statistical analysis of soluble protein ratio

E. coli

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

                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

• 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

Alternative Expression Systems

Baculovirus expression system

Bright filed UV merged

- EGFP expressed in baculovirus transfected insect cell

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

                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

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

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

Self-cleavage of fusion protein in vivo using TEV protease to yield native protein

• 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'-

In vivo cleavage of fusion proteins.

TEVP intracellular processing system

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

all six vectors successfully carried out intracellular cleavage

TEVP intracellular processing system

• 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