Transcript 投影片 1

Recombinant Protein Production
- Introduction to Expression Systems
- Core Facility of Recombinant Protein Production,
National Research Program for Genomic Medicine
Recombinant Protein Production
-Why?
• over-expression to get enough amount
• easy purification
-Application
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functional studies
structural studies
vaccine/antigen/antibodies
therapeutic drug
industrial enzymes for reaction
Application: Drug Discovery
Application: therapeutic proteins
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Actimmune (If g)
Activase (TPA)
BeneFix (F IX)
Betaseron (If b)
Humulin
Novolin
Pegademase (AD)
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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
Principle in Protein Production
Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications
Protein Expression Bottleneck
DNA
Cloning
Expression
Purification
Enzymology
Crystallography
• 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
l Inefficient transcription
No or little protein synthesized
u Promoter choice and design
l Inefficient translation
No or little protein synthesized
u Codon usage
u Transcript stability
u Transcript secondary structure
l Inefficient folding (cytoplasmic or periplasmic)
Aggregation or degradation
u Improper secondary, tertiary or quaternary structure formation
u Inefficient or improper disulfide bridge formation
u Inefficient isomerization of peptidyl-prolyl bonds
l Inefficient membrane insertion/translocation
l Toxicity
Cell death
Aggregation or degradation
Protein Expression and Purification
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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:
Gene of Interest
+
Expression Vector
Expression of Fusion Protein
Recombinant
Vector
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
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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
mammalian 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
promoters, 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
protein inside the cell
• 2 routes of transformation
– CaCl2 + cold shock
– Electroporation
• Typical transformation rate is 1 in 10,000 cells (not very
efficient) 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
Advantages
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Grow quickly (8 hrs to produce
protein)
High yields (50-500 mg/L)
Low cost of media (simple media
constituents)
Low fermentor costs
Disadvantages
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Difficulty expressing large proteins
(>50 kD)
No glycosylation or signal peptide
removal
Eukaryotic proteins are sometimes
toxic
Can’t handle S-S rich proteins
Cloning & Transforming in Yeast
Cells
Pichia pastoris
Pichia Pastoris
• Yeast are single celled eukaryotes
• Behave like bacteria, but have key advantages of
eukaryotes
• P. 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)
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
linearized (cut open so it isn’t circular)
• Double 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
Pichia Systems
Advantages
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Grow quickly (8 hrs to produce
protein)
Very high yields (50-5000 mg/L)
Low cost of media (simple media
constituents)
Low fermentor costs
More advantages
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Can express large proteins (>50 kD)
Glycosylation & signal peptide removal
Has chaperonins to help fold “tough”
prtns
Can handle S-S rich proteins
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
Transfer vector
Cloned gene
5’
3’
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x
Cloned gene
5’
3’
Polyhedrin gene
AcMNPV DNA
Recombinant
AcMNPV DNA
Baculovirus Systems
Disadvantages
• 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
Advantages
• Can express large proteins
(>50 kD)
• Correct glycosylation &
signal peptide removal
• Has chaperonins to help fold
“tough” prtns
• Very high yields, cheap
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
Transfect
dfhr- cells
Grow in
Nucleoside
Free medium
Culture a
Colony of
cells
Grow in
0.05 uM Mtx
Culture a
Colony of
cells
Methotrexate (MTX) Selection
Grow in
0.25 uM Mtx
Culture a
Colony of
cells
Grow in
5.0 uM Mtx
Culture a
Colony of
cells
Foreign gene
expressed in
high level in
CHO cells
Mammalian Systems
Disadvantages
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Selection takes time (weeks for set-up) •
Cell culture is only sustainable for
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limited period of time
Set-up is very time consuming, costly, •
modest yields
Advantages
Can express large proteins (>50 kD)
Correct glycosylation & signal peptide
removal, generates authentic proteins
Has chaperonins to help fold “tough”
prtns
Conclusion
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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
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E. coli
Baculovirus
Yeast
Cell-free
Mammalian cell
( not open for service)
Expresssion Systems
SYSTEMS
Advantages
Disadvantages
E. coli
•Parallel cloning
•Fast
•Ease of use
•Low cost
•Poor expression
•Low solubility
•Lacking post-translational modifications
Cell-free
•Low protein yield
•Faster
•Expensive
•Skips cell transformation, growing,
•Tricky to optimize the lysate
and lysis
•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 •Lower protein yield
proteins
•Expensive
E. coli
- the most popular expression system
E. coli Expression System
-challenge
• poorly expressed
• protein insoluble- inclusion bodies
• expressed and soluble: 20-30%
-improvement
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growth condition (e.g. temperature)
codon usage
host strain
fusion to carrier protein
E. Coli Expression System
parallel screening for soluble proteins
1. Increase the expression level and solubility of
target protein with protein tags.
Rationale
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
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 Expression System - Modified version
EcoR I
Promotor
Xho I
Terminator
Target
Fusion tag
His*6
Thrombin
FXa
Protein
His*6
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
E. coli
技術比較說明
融合蛋白質的選擇類似，主要是cloning的差別
Hammarström et al. Protein Science (2002), 11:313–321
他人已使用商品化的策略；Gateway Technology (Invitrogen)
PCR
Ligation
Donor
vector
Purify
plasmid
Co-transformation
我們使用Sticky-end PCR的方法，不必經過Sub-cloning
即可parallel cloning
PCR
Denature
Re-nature
E. coli
E. Coli Expression System Summary
• The method introduces sticky-end to target genes,
without using restriction enzymes.
• Well-induced and highly soluble recombinant proteins :
80% success
Alternative Expression Systems
Baculovirus expression system
- EGFP expressed in baculovirus transfected insect cell
Bright filed
UV
merged
Cell-free expression system
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6
1: Negative control
2: Positive control (GFP)
3: Hpps component II
4: Hyaluronan synthase
5: Rubber prenyl transferase
6: Marker
Yeast expression system
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2
3
1: Marker
2: N3D TPL-2 using horseshoe
crab signal peptide
3: N3D TPL-2 using pichia
signal peptide
服務項目介紹
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
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
14,000
http://proteome.sinica.edu.tw/prod_services_01.asp
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
Flow chart of protein production
Service
Requested
Parallel Cloning
Expression test in E. coli
additional charge
standard
Insoluble / posttranslational modification required
Soluble
Yeast system
Baculovirus system
in vitro expression systems
Protein Purification
Protease cleavage to
remove tag
Self-cleavage of fusion protein in vivo
using TEV protease to yield native protein
-challenge to fusion protein method
separation of passenger target protein from the fusion carrier
• fusion carriers cannot be processed by proteolysis
• cleaved products aggregate immediately
• cleaved products contain extraneous a.a. residues
-our approach
• TEVP intracellular processing system
tobacco etch virus protease (TEVP)
-Glu(P6)-P5-P4-Tyr(P3)-P2-Gln(P1)- -P1'-
TEVP intracellular processing system
In vivo cleavage of fusion proteins.
TEVP intracellular processing system
Different amino acid residues at the P1' position
- more effective than an intermolecular enzymatic reaction
- even with Pro in the P1' position
TEVP intracellular processing system
all six vectors successfully carried out
intracellular cleavage
TEVP intracellular processing system Summary
• 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