Recombinant Protein Expression Protocols PDF

Protocols Dr. Mahmoud Saleh For Biotechnology students Associate professor Faculty of Science, Cairo University Department of Botany and Microbiology Lab 3. Induction of Recombinant Protein Expression Recombinant protein expressi...

Protocols Dr. Mahmoud Saleh For Biotechnology students Associate professor Faculty of Science, Cairo University Department of Botany and Microbiology Lab 3. Induction of Recombinant Protein Expression Recombinant protein expression: Escherichia coli is the host of choice for the first attempt at recombinant protein production, regardless of the original source. Recombinant protein expression is performed in E. coli strains BL21 (DE3) pLysS + RIL. IPTG induction protocol: One of the most used E. coli expression systems relies on the inducible T7 RNA polymerase because this system obtains high yields of recombinant proteins. The coding sequence of the T7 RNA polymerase is inserted into the bacterial chromosome under the control of the inducible lac UV5 operon and is transcribed by the endogenous E. coli polymerase. The lac repressor protein (LacI) regulates access to the T7 RNA polymerase coding sequence by binding to the lac UV5 operon. Protein expression induction is triggered by the addition of the inducer, isopropyl-β -D-1-thiogalactopyranoside (IPTG), which is a structural nonmetabolizable analogue of allolactose. IPTG is currently the most efficient method to induce promoter expression. The T7 RNA polymerase produced after induction specifically transcribes the coding sequence of the protein of interest that is inserted into the expression plasmid under the control of the T7 promoter. Moreover, access to the plasmidic T7 promoter can be regulated by the lacI repressor when the T7 promoter is fused with the lac operator (T7 lac promoter). Protocol: 1. From a relatively fresh plate (