1. Chapter 8: Expression and Modification of Recombinant Proteins
Prokaryotic expression system
Eukaryotic expression system
Yeast expression
Insect expression system
Mammalian expression system

2. References Chapter 11 and 13 (Essentials of Molecular Biology)
Chapter 10 (An Introduction to Genetic Engineering)

3. I. Prokaryotic Expression System
Friday, April 21, 2017
I. Prokaryotic Expression System
The red boxes represent exons, the blue boxes represent the introns, and the grey boxes represent the 5’ and 3’ UTRs
the gene of eucaryotic includes additional sequences that lie within the coding region, The sequences of DNA comprising an interrupted gene are
divided into the two categories,
• The exons are the sequences represented in the mature RNA. By definition, a gene starts and ends with exons, corresponding to the 5' and 3' ends of the RNA.
• The introns are the intervening sequences that are removed when the primary transcript is processed to give the mature RNA.
How do you express this gene in bacteria?
Insert only the ORF:
into an expression vector
that contains prokaryotic transcriptional and
translational regulatory sequences

4. Six Step Process Isolation of gene of interest
Introduction of gene to expression vector
Transformation into host cells
Growth of cells through fermentation
Isolation & purification of protein
Formulation of protein product

5. 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)

6. Fundamentals of Gene Expression
Prokaryotic and eukaryotic promoters and translation signals are different...they are not exchangeable
You therefore can’t simply put a eukaryotic promoter into bacteria and expect it to function

7. The genetic code is identical for most bacterial and eukaryotic mRNAs.
Processing also presents a problem for bacterial expression of human mRNAs.
The eukaryotic ORF must be cloned into the expression vector, not the genomic copy
The genetic code is identical for most bacterial and eukaryotic mRNAs.
Although the code is the same, expression levels can be affected by codon frequency, which varies between organisms and transcripts
Post-translational modifications can be important for protein function
Those modifications might not occur in bacteria. The solution…try expressing in a eukaryotic expression system (viral, baculovirus, yeast)

8. Prokaryotic Expression
Pros
Convenient/Easy
Produce and purify protein using the least expensive and easiest reagents and equipment
Best for large scale production of protein
Cons
lack many of the immunogenic properties
3D “Native” conformation
Lack PTMs (Post translational modifications) needed for specific activity

9. Which Vector? Tags标签 Promoters
arabinose systems (pBAD), phage T7 (pET), Trc/Tac promoters, λ PL or PR
Tags标签
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

10. Gene ( with / without stop codon )
Fusion Protein
Gene ( with / without stop codon )
T7 Promoter
RBS
ATG
TAG
lac Operator
peptide tag: V5 / poly-(his)
mutiple cloning site (mcs)
peptide tag: V5 / poly-(his)

11. A Generic Vector

12. II. Eukaryotic Expression System
Yeast expression
Insect expression system
Mammalian expression system

13. 1. Yeast Expression
􀂄 Pros
Easier and less expensive than higher eukaryotic cells
Rapid growth on inexpensive media
Ideal for large-scale production of heterologous proteins
Often comfortable with genetic manipulation
Exhibit near-native conformation, PTMs, processing
Well-defined secretory pathways for extracellular export of the recombinant gene product
Usually safe to use
Cons
Lack some PTMs required for specific activities
A little more expensive than prokaryote systems
Often lack mechanisms for proper folding for some eukaryote proteins

14. Yeast Expression Vector
Comments for pPIC9K: 9276 nucleotides
5´ AOX1 promoter fragment: bases 1-948
5´ AOX1 primer site: bases
a-Factor secretion signal(s): bases
a-Factor primer site: bases
Multiple Cloning Site: bases
3´ AOX1 primer site: bases
3´ AOX1 transcription termination (TT):
bases
HIS4 ORF: bases
Kanamycin resistance gene: bases
3´ AOX1 fragment: bases
pBR322 origin: bases
Ampicillin resistance gene: bases
Pichia pastoris

15. Yeast are single celled eukaryotes
Behave like bacteria, but have key advantages of eukaryotes
P. pastoris is a methylotrophic甲醇为营养的yeast that can use methanol甲醇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)

16. Cloning in Yeast Cells
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

17. Cloning 3 1 2 4

18. 2. Insect Expression Pros Cons
Produce proteins that has PTMs similar to mammalian systems
Often properly folded and functional
Ideal for producing moderate to high levels of eukaryotic proteins for structure-function assays
Cons
Expensive
Sometimes proteins are not correctly folded
Often not stable

19. 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

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

21. Baculovirus

22. BacVirus Expression System
pIEx™Insect Cell Expression Plasmids (Novagen)
Bac-N-Blue™Baculovirus Expression System (Invitrogen)

23. 3. Mammalian Expression Pros
Produce protein in the most native and active form
Have required PTM machinery to produce active and useable protein used in mammals
Cons
Expensive
Unstable
Low yield and difficulties in purifying recombinant proteins
Limitations on the mechanisms of protein expression induction
Almost always have over expression

24. HeLa cells in culture

25. HeLa cells from the Nikon microscope web site
HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s. They have been utilized for many purposes, including the development of a polio vaccine, the pursuit of a cure for diseases such as leukemia and cancer, and the study of the
cellular effects of drugs and radiation.

26. JONATHON PINES REGULATION OF MITOSIS IN MAMMALIAN CELLS
HeLa Human cells
JONATHON PINES REGULATION OF MITOSIS IN MAMMALIAN CELLS
Mitotic HeLa cell stained with anti-Cks1 (red), anti-tubulin (yellow) and DAPI (blue)

27. HeLa cells as you will see them
Expression of (A) b-galactosidase and B green fluorescent protein in HeLa cells. Cells were transfected in 6-well plates. Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection.

28. 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 baculovirusor杆病毒 mammalian cell culture
High yields, low cost? Choose E. coli
Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote

29. 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

30. 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

31. 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

32. 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

33. 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

34. Mammalian Expression Systems
Comments for pcDNA™4/TO : 5078 nucleotides
CMV promoter: bases
TATA box: bases
Tetracycline operator (2X TetO2) sequences: bases
CMV forward priming site: bases
Multiple cloning site: bases
BGH reverse priming site: bases
BGH polyadenylation sequence: bases
f1 origin: bases
SV40 promoter and origin: bases
EM-7 promoter: bases
Zeocin™ resistance gene: bases
SV40 early polyadenylation sequence: bases
pUC origin: bases
bla promoter: bases (complementary strand)
Ampicillin (bla) resistance gene: bases (complementary strand)

35. 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

36. Summary
Friday, April 21, 2017
The mechanism of regulation of gene expression in prokaryotes is greatly different from eukaryotes
Regulation of gene expression in prokaryotes is mainly for environmental adaptation
Regulation of gene expression in eukaryotes is for cell growth, differentiation and development
There are four recombinant protein expression systems: bacteria (E.coli), yeast, insect and mammalian cells. Choose by purpose