This document discusses recombinant protein expression in different systems including bacterial, yeast, insect, mammalian and plant systems. It provides details on the process of recombinant protein expression which involves isolation of mRNA, cDNA synthesis, cloning into an expression vector, transformation into a host cell, protein expression and recovery. The advantages and disadvantages of expressing proteins in different cellular compartments like cytoplasm, periplasm and culture medium are also mentioned. Quality control methods like SDS-PAGE and western blotting are discussed.

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