RECOMBINANT PROTEIN EXPRESSION

BYKARIUKI M. SAMUEL

ENOS KAMANI

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.

INTRODUCTION CONT’

Bacterial expression

• Isolation of total or poly A tail RNA from cells or tissue

• First cDNA

Bacterial expression cont’

• Addition of oligonucleotide plugs

Bacterial expression cont’

• PCR amplification of cDNA

Bacterial expression cont’

• Directional ligation into appropriate vectorPeriplasmic export extracellular export

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

Bacterial expression cont’

• Protein expression recovery sites in E. coli-cytoplasmic, periplasmic, medium

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

Bacterial expression cont’

• Advantages and disadvantages in the protein production into each compartment.

Bacterial expression cont’

• Purification-affinity chromatography• Quality control:• Acrylamide gel, • SDS-PAGE, • Western blot. 12

3 4

Yeast expression

• Same protocol as bacteria but the host is yeast cells where your protein is harvested

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

Insect expression

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

Mammalian expression

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

Expression in plants

Conclusion

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

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