1. Recombinant Protein Production
Introduction to Expression Systems
Core Facility of Recombinant Protein Production,
National Research Program for Genomic Medicine

2. Recombinant Protein Production
-Why?
over-expression to get enough amount
easy purification
-Application
functional studies
structural studies
vaccine/antigen/antibodies
therapeutic drug
industrial enzymes for reaction

3. Application: Drug Discovery

4. Application: therapeutic proteins
Actimmune (If g)
Activase (TPA)
BeneFix (F IX)
Betaseron (If b)
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 (

5. Application: structural genomics
Bioinformatics

6. Principle in Protein Production
Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications

7. Protein Expression Bottleneck
Protein Biochemistry
soluble, purifiable protein
Enzymology
soluble, active protein
mg of protein
Crystallography
soluble, crystallizable protein
5-100 mg of protein

8. Bottlenecks to efficient protein expression in E. coli
Inefficient transcription
No or little protein synthesized u
Promoter choice and design l
Inefficient translation
No or little protein synthesized u
Codon usage
Transcript stability
Transcript secondary structure u u l
Inefficient folding (cytoplasmic or periplasmic)
Aggregation or degradation u
Improper secondary, tertiary or quaternary structure formation
Inefficient or improper disulfide bridge formation
Inefficient isomerization of peptidyl-prolyl bonds u u l
Inefficient membrane insertion/translocation
Aggregation or degradation l
Toxicity
Cell death

9. 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

10. + Cloning and expression of target gene: Expression of Fusion Protein
Gene of Interest
Recombinant
Vector
Expression Vector
Expression of Fusion Protein

11. 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)

12. Cloning (Details)

13. Cloning (Details)
protein

14. Recombinant Protein Expression Systems
Escherichia coli
Other bacteria
Pichia pastoris
Other yeast
Baculovirus
Animal cell culture
Plants
Sheep/cows/humans
Cell free
Polyhedra

15. 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

16. 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

17. 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)

18. 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

19. A Generic Vector

20. 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

21. Gene Introduction (Bacteria)

22. Bacterial Transformation

23. 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

24. Electroporator
25 microfarads = 2500 V
@ 200 ohms for 5 ms

25. 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

26. Bacterial Systems Advantages Disadvantages
Grow quickly (8 hrs to produce protein)
High yields ( 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

27. Cloning & Transforming in Yeast Cells
Pichia pastoris

28. 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)

29. Pichia Cloning

30. 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

31. Pichia Systems Advantages More advantages
Grow quickly (8 hrs to produce protein)
Very high yields ( 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

32. Baculovirus Expression

33. 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

34. Baculovirus Expression
~12 days

35. Baculovirus (AcMNPV) Cloning Process5’ 3’
Transfer vector
Polyhedrin gene x
Cloned gene
Cloned gene 5’ 3’
AcMNPV DNA
Recombinant
AcMNPV DNA

36. Baculovirus Systems Disadvantages Advantages
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

37. Mammalian Expression Systems

38. 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

39. 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

40. 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

41. 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

42. Mammalian Systems Disadvantages Advantages
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

43. 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

44. National Research Program for Genomic Medicine
Core Facility of Recombinant Protein Production重組蛋白質生產核心設施 D1

45. Expression systems E. coli Baculovirus Yeast Cell-free
Mammalian cell ( not open for service)

46. 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
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

47. E. coli - the most popular expression system

48. E. coli Expression System
-challenge
poorly expressed
protein insoluble- inclusion bodies
expressed and soluble: 20-30%
-improvement
growth condition (e.g. temperature)
codon usage
host strain
fusion to carrier protein

49. parallel screening for soluble proteins
E. Coli Expression System
parallel screening for soluble proteins
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:

50. E. coli
Sticky-end PCR

51. E. coli
Parallel Gene Cloning

52. Parallel screening for soluble protein
E. coli
Parallel screening for soluble protein

53. Statistical analysis of soluble protein ratio
E. coli
Statistical analysis of soluble protein ratio

54. E. coli Expression System - Modified version
EcoR I
Xho I
Promotor
Fusion tag
Thrombin
His*6
FXa
Target
Protein
Terminator
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 I
Promotor
Fusion tag
Thrombin
His*6
FXa
Target
Protein
Terminator

55. 技術比較說明 融合蛋白質的選擇類似，主要是cloning的差別
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)
Donor vector
Purify plasmid
PCR
Ligation
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

56. 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

57. Alternative Expression Systems

58. Baculovirus expression system
- EGFP expressed in baculovirus transfected insect cell
Bright filed UV merged

59. Cell-free expression system Yeast expression system
1: Negative control
2: Positive control (GFP)
3: Hpps component II
4: Hyaluronan synthase
5: Rubber prenyl transferase
6: Marker
1: Marker
2: N3D TPL-2 using horseshoe crab signal peptide
3: N3D TPL-2 using pichia signal peptide

60. 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
(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

61. 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

62. 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

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

64. -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'-

65. In vivo cleavage of fusion proteins.
TEVP intracellular processing system
In vivo cleavage of fusion proteins.

66. Different amino acid residues at the P1' position
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

67. all six vectors successfully carried out intracellular cleavage
TEVP intracellular processing system
all six vectors successfully carried out intracellular cleavage

68. 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