This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.

1. Strategies For the Production of
Recombinant Protein in E.coli
Gopal Jee Gopal, PhD(JNU)
Assistant Professor
C.G.Bhakta Institute of Biotechnology
Uka Tarsadia University, Bardoli

2. Why do we need protein?
Proteins are used as therapeutics e.g. growth
hormone, Insulin, Vaccines etc.
Used As enzymes for various purposes e.g. Restriction
enzymes, polymerases, Proteases, lipases, etc.
For research : We need good amount of protein
with high purity to characterize that protein.
( Genome/gene sequencing is very easier than
Characterization of protein encoded by that
genome/gene.)

3. How can we get desired amount of
protein with good quality?
1. Purification from their endogenous source.
Serious limitations: Quantity, Purity, consistency etc.
or
2. Applying recombinant DNA technology/Molecular
cloning.

4. Therapeutic Recombinant protein
T A. Brawn

5. Overview of recombinant DNA technology
Bacterial cell
Bacterial
chromosome Plasmid
Gene of interest
DNA containing
gene of interest
Isolate plasmid.
Enzymatically cleave DNA into fragments.
Isolate fragment
with the gene of
interest.
Ligate gene into plasmid.
Transform bacteria with ligated plasmid
Culture bacteria.
Harvest copies of gene to insert into
plants or animals
Harvest the protein encoded in the cloned gene.

6. Players of the gene cloning
• Passenger gene (Insert).
• Vehicle DNA molecule (Vector).
(Cloning and Expression)
• Enzymes.
• Host organisms e.g. Bacteria, yeast,
Mammal, plant etc.

7. Why E.Coli is preferred choice?
Easy to culture
Very short life cycle
Cloning is technically easier
Well known Genetics

8. Limitations of normal E.coli host
 Difficult or little expression of foreign gene
 Post translational modification does not
occur.
 No disulphide bond formation in cytoplasm.
 Inclusion body formation on over
expression.

9. What are the strategies to get rProtein
in good amount and of good quality
B. By changing the vector:
A. By changing host strain:
C. By changing culture parameters
D. By co-expression:
E. By altering the gene sequence:

10. By changing host
pET system: eg. pET 28a: Common choice, small tag but robust.
Histidine tag
T7 promoter

11. BL21(DE3)
BL 21 (DE3):
E.coli strain having T7 polymerase
gene inserted in its genome.
Protease defficient:Lon and OmpT
Limitations: Expression is leaky

12. Expression of gene cloned under T7
promoter.

13. BL21 (DE3) pLys.
Lysozyme coding plasmid
Very less leaky expression
May be used for toxic protein expression
too.
Chloramphenicol resistance(pACYC)

14. How to overcome effect of codon
Biasness
Expression of foreign gene in heterologous host encounters
with the problem of codon bias in expression host.
Due to this bias, it may be difficult to express recombinant
protein or may result in a truncated protein.

15. Codon Human Drosophila E.coli
Arginine
AGA 22% 10% 1%
AGG 23% 6% 1%
CGA 10 8 4
CGC 22 49 39
CGA 14 09 4
CGU 09 18 49
Total No of
arginine
codons
2403 506 149
Serine codons
AGT 10 1 3
AGC 34 23 20
TCG 9 17 4
TCA 5 2 2
TCT 13 9 34
TCC 28 48 27
Attwood and Parry Smith ,1999.

16. Codon Plus(RIL/RP)
Overcome the limitation of codon biasness of
heterologous gene.
BL21-CodonPlus-RIL cells contain extra copies of the
argU,ileY, and leuW tRNA genes, which recognize the
AGA/AGG,AUA, and CUA codons. Overcome the
problem of AT rich gene.
BL21-CodonPlus-RP competent cells contain extra
copies of the tRNA gene argU, which recognizes the
AGA and AGG arginine codons, and the tRNA gene
proL, which recognizes the proline codon, CCC. This
strain overcome the bias of GC rich genome.
Stratagene

17. Rosetta strain(Novagen)
Supply tRNAs gene for the codons AUA,
AGG, AGA, CUA, CCC, and GGA on a
compatible chloramphenicol-resistant
plasmid, pRARE.
Can be used for both AT or GC rich
genome bias.

18. BL21-C43 cell
• Self Mutant E.coli strain, genetics like
BL21(DE3).
• Can express toxic and globular protein(Bruno
Miroux and John E. Walker, 1996).
• overcoming the problem of plasmid instability
during toxic recombinant protein expression.
(Laurence Dumon-Seignovert etal., 2004)
• Supplied by Lucigen

19. Difference in sequence that cause
better expression of toxic gene or
globular gene

20. Lemo 21(DE3)
Derivative of BL21(DE3)
Supplied by NEB
T7 poymerase cloned under titrable rhamnose promoter
Can be used for expression of toxic and globular protein.

21. ArcticExpress E.coli
Efficient folding of Protein even at low temperature
This genetically engineered strain co-express cold-
adapted chaperonins Cpn10 and Cpn60.
Supplied by Agilent Technolgy.

22. BL21 star
Supplied by invitrogen
rne131 gene mutation confers high stability of mRNA.
rne131 gene encodes RNase which degrades mRNA.

23. Expression of protein which require
disulphide bond.
E.Coli cytoplasm has reducing environment so it doesnot
allow formation Of disulphide bonds.
Periplasm of E.coli has oxidising environment so here
Disulphide bond formation takes place.
Cloning of desired gene into a Vector which has signal peptide
coding sequence before MCS is used to transport desired protein
into periplasm if disulphide bond formation is required.
But cytoplasmic expression results in high
yields than periplasmic expression.

24. Origami/Shuffle strain(Novagen/NEB)
• Having oxidising environment in host cytoplasm.
• Mutations in both the thioredoxin reductase
(trxB) and glutathione reductase (gor) genes.
• Shuffle strain expresses disulphide bond
isomerase(dsbC) that helps in proper disulphide
bond formation.
* These E.coli strains allows Disulphide bond in the
cytoplasm.

25. Different types of shuffle strain
SHuffle Express Competent E. Coli:
Protease Deficient B Strains
SHuffle T7 Express E. coli: Protease Deficient
B StrainsT7 Expression
SHuffle T7 Express lysY Competent E. Coli:
Protease Deficient B Strains T7 Expression ,
Tight control/expression of toxic proteins

26. Glycosylation of recombinant protein in E.coli
Glycosylated recombinant protein has also been produced
in genetically engineered E. coli host using pgl operon of
Campylobacter jejuni by Michael W et al.(2002).
This glycosylation is different than eukaryotic protein
glycosylation.
Valderrama-Rincon et al. Successfully engineered an
E. coli strain and got N-glycosylation of eukaryotic
recombinant protein.
These strains are not commercially available.

28. Changing the Vector
pMAL- Vector: (NEB)
Maltose Binding protein tag.
44kDa tag
 Tac promoter:(hybrid of tryptophan
and Lac).
Increase solubility of tagged
protein
(diGuan et al., 1988).

29.  NUS (N utilisation substance)tag has high potential of
protein solubility
(Harrison 2000, Davis 1998).
e.g. pET44 and pET43 vector(Novagen)
pHAT vector(Clontech) is better than pET vector.
 Nonadjacent histidine
 Lower overall charge than 6 adjacent histidine
Solubility similar to wild type protein

30. pHAT vector

31. pGeX system
Supplied by GE healthcare
Glutathione S-Transferase tag (GST).
25KDa tag
Increase solubility of tagged protein.
(Smith and Johnson, 1988)

33. pQE vector system
Supplied by Qiagen
Gene is cloned under T5 promoter which is recognized by
E.coli polymerase.
The host for expression of gene cloned in this vector is either M15 or
SG13009 E.coli strain. These strains possess pREP4 plasmid.
This plasmid encodes lac repressor which ensures tight regulation of
expression.

34. pSUMO
Solubility of conjugated protein increases.
 Precise deletion of tag is possible.
 Derivative of this vector is available for different
antibiotic resistance and of dual expression also.
Supplied by Life sensor inc.

36. RE/MCS site of Vector
pET28a-
pMAL-p/c 5X:
pGEX-
pQE30-
BamHI
EcoRI
SmaI
SalI
XhoI
NotI
NcoI
NdeI
NheI
HindIII
SacI
XmnI
NdeI
NcoI
NotI
EcoRV
NotI
SalI
BamHI
EcoRI
SbfI
BamHI
EcoRI
SalI
EagI
XhoI

37. Physical or chemical agents
1. Culture of recombinant host at low
temperature.
2. Ethanol addition: Steczko et al.1991
3. Heat shock: Makrides,1996
4. Salt addition: Dinnbier et al.1988
5. Benzyl alcohol addition: de Marco et al.,2005
6. Osmolyte addition: de Marco et al.2005, Diamant et
al.2003 and Schultz et al.2007
7. Trehalose : Schultz et al.2007

38. Co-expression
 Desired gene co-expressed with different
combination of chaperone showed promising
results.(Ario de Marco,2007).
 Co-expression of interacting partner of a complex.

39. Changing the gene sequence
Removal of signal pepetide coding sequences from ORF
 Removal of highly hydrophobic stretch from protein.
 Optimisation and synthesis of gene before cloning.
 Changing few rare codons using site directed mutagenesis.

40. Conclusion
Gopal GJ and Kumar A, 2013

41. References
Gopal GJ, kumar A (2013) Strategies For The Production of
Recombinant Protein in Escherichia.Coli. Protein J. 32: 419-425.

42. Fellowships for candidate Pursuing PhD in Life
Science and other science stream
1. CSIR/UGC- a)NET-LS b) JRF c)SPM
2. ICMR- a)JRF b)for project
3. DBT a) b)
4. Prime minister’s fellowship scheme( Fellowship:Rs.55K to 72K PM)
5. National institutes have their own fellowship equivalent to JRF
6. Central Universities have their own fellowship:
7. The Research Fellowship in Sciences for meritorious students (RFSMS) .
8. Rajiv Gandhi fellowship for Sc/St
9. Maulana Azad Fellowship for minority
10. Inspire fellowship: For University topper

43. Thank You
For Attention
Send your feedback at
gopaljigopal@rediffmail.com
Gopal.jee@utu.ac.in
or at 9558880617, 8160245501