The Expression Of GABA_B Receptor In E.Coli, Purification And Renaturation

GABA is the most important inhibitory neurotransmitter in central nervoussystem of breastfeed mammal. GABA_B receptors are metabotropic receptor, alsonamed G protein-linked receptor. GABA_B receptors are linked via G-proteins to thecell’s conductance to Ca²⁺ and potassium channels to realize physiology function.In this research the inclusion body was successfully expressed in BL21（DE3）which contained pET28c plasmid. The gene was expressed as a fusion proteinGABA_BR with a His-tag at its N terminal to use further affinity purification. Itsoptimum concentration of IPTG is 1mM ,optimum induced time is 3h and optimumtemperature is 37℃. The recombinant E.coli was induced with 1mM IPTG for 3h in 37℃and 1mL culture medium could produce 70μg GABA_BR protein, but the protein wasexpressed with inclusion bodies. The inclusion body was washing with preliminarypurification, then was dissolved by 5M Gu.Hcl and 8M urea .GABA_BR protein wasthen purified by Sephacryl S-200 column under denaturing conditions （5M Gu.Hcl）,the protein assay is to 95%.The GABA_BR protein was refolded by dilution, and about70.6% protein was refolded. The GABA_BR protein was then refolded by stepwisedialysis with the Gu.Hcl concentration（5M Gu.Hcl） in the base dialysis buffer reducedas follows 4M-3M-2M-1M-0M.After stepwise dialysis in 4℃, about 26% protein wasrefolded. The GABA_B protein was also successfully refolded while it was immobilizedon Ni-NTA, by washing it using refolding buffer with urea concentration as follows6M-5M-4M-3M-2M-1M-0M under denaturing conditions （8M urea）.About 40%proteins were refolded. This method realized purification and renaturation at the sametime.