RECOMBINANT PROTEIN EXPRESSION BY KARIUKI 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.
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 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• 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
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