a seminar presentation paper

1. RECOMBINANT PROTEIN
EXPRESSION
BY
KARIUKI M. SAMUEL
ENOS KAMANI

2. INTRODUCTION
• RPE is a subcomponent of gene expression
which involves translation of the gene into
polypeptide chains and folding into proteins
• Used in esp. enzyme production, insulin,
endonucleases, DNA pol, reverse T.
• Classified according to expression systems;
bacterial, yeast, insect, mammalian.

3. INTRODUCTION CONT’

4. Bacterial expression
• Isolation of total or poly A tail RNA from cells
or tissue
• First cDNA

5. Bacterial expression cont’
• Addition of oligonucleotide plugs

6. Bacterial expression cont’
• PCR amplification of cDNA

7. Bacterial expression cont’
• Directional ligation into appropriate vector
Periplasmic export extracellular export

8. Bacterial expression cont’
• Amplification of the primary library into a
bacteria of choice, E. coli, B. subtilis.
• Transformation done through heat shock,
electroporation.
Heat shock transformation

9. Bacterial expression cont’
• Protein expression recovery sites in E. coli-
cytoplasmic, periplasmic, medium

10. Bacteria expression cont’
• Harvesting done by
• Chemical
• Enzymatic e.g lysozymes
• Biochemical e.g osmotic shock
• Physical e.g. sonication
• Mutant E. coli strains, L-forms

11. Bacterial expression cont’
• Advantages and disadvantages in the protein
production into each compartment.

12. Bacterial expression cont’
• Purification-affinity chromatography
• Quality control:
• Acrylamide gel,
• SDS-PAGE,
• Western blot. 2 1
3 4

13. Yeast expression
• Same protocol as bacteria but the host is yeast
cells where your protein is harvested

14. Expression in yeast cont’
• Genes synthesis
• cDNA cloning in Yeast expression factor and
tagging
• Transformation in yeast cells
• Confirmation of pos. trans-formants by PCR
• Affinity purification
• SDS-PAGE/ western blot

15. Insect expression

16. Insect expression cont’
• Gene synthesis
• cDNA cloning in baculovirus expression vector
• Recombinant virus generation and
recombinant protein in bacmid
• Transfection of insects cells with recombinant
bacmid
• Generation of virus stock in insect cells
• Quality control, SDS-PAGE/ western blot

17. Mammalian expression

18. Mammalian expression
• Gene synthesis
• cDNA cloning into an expression factor
• Transfection of cells with expression vector
• Evaluation of expression by western blotting
• Bio-reactor production
• Harvesting
• Affinity chromatography
• Quality control, SDS-PAGE/ western blot

19. Expression in plants

20. Conclusion

21. References
• Artes Biotechnology Langenfeld, Germany, http://www.artes-
biotechnology.com/expres/hansenula/hansenula01.jsp
• Dual SystemsBiotech, Schleren, Switzerland.
http://www.dualsystems.com/products/protein-expression/expression-vectors-
for-escherichia-coli.html
• Genscript, the biology GRO, Piscataway, USA.
http://www.genscript.com/custom_protein_baculovirus_insect_expression.html
• Joan L. &J. Foster (2009) Microbiology, an evolving science. W.W. Norton, New
York, USA 2nd ed.
• Park, S. J. and Lee, S. Y., "Efficient recovery of secretory recombinant protein from
protease negative mutant Eshcherichia coli" Biotechnol. Techniques, 64(12):4897-
4903 (1998)
• Tokyo Future Style Inc.
http://www.tokyofuturestyle.com/english/products/preventec/preventec_t.php

22. Merry-xmas