Prokaryotic Expression, Purification,Characterization And Preparation Of Polyclonal Antibody For Malonyl/Acetyl Transacylase Of Xinong Saanen Goat

Acetyl-CoA and malonyl-CoA transacylases (MAT) which is one domain of FASN is the key enzyme for short-chain fatty acids synthasis in vivo. It contains the ability to terminate the carbon chain extension of fatty acid to release the short-chain fatty acid. Because commercial specific antibodies against MAT protein are unavailable now, so it is necessary to prepare the antibodies against MAT to further study the biological function of MAT in Fatty Acid Metabolism. In our study, MAT was amplified by PCR from the pAdTrack-CMV-MAT plasmid. We sub-cloned MAT gene of goat into the vector pET32a (+) to construct prokaryotic expression vector pET32a(+)-MAT, then transformed it into E. coli BL21(DE3) competent cells. Recombinant protein was purified by Ni-NTA column and was then refolded by gradient dialysis. We then analysed the enzyme activity of refolded MAT by in vitro enzymatic reaction. The products was identifed by high performance liquid chromatography (HPLC) method. Recombinant protein emulsified with adjuvant was used as antigen to immune rabbit to prepare polyclonal antibody. iELISA analysis and Western blot were used to detect the titer and the specificity of the polyclonal antibody. The main results of this research are as follows:(1) The MAT gene was coloned by PCR with the length of981bp, the PCR products was cloned into the prokaryotic expression vector pET32a(+). We then transformed pET32a(+)-MAT vector into E. coli BL21(DE3). Recombinant protein expressed successfully. Western blot showed that the recombinant protein has6×His tag which can be used for purification.(2) We optimized the expression conditions of prokaryotic vector pET32a(+)-MAT expressed in bacteria BL21(DE3), that is:37℃,0.5mmol/L of IPTG induction for6h; the target protein was mainly expressed in the form of fusion protein under these conditions.(3) We purified the recombinant protein from inclusion body lysates using Ni-Agrose affinity purification method. The recombinant MAT was refolded by gradient urea dialysis, then concentrated by PEG20000. The concentration of the recombinant protein was0.5mg/mL by BCA method.(4) The enzymatic activity assay of the purified recombinant protein showed that the refolded protein retained the transferase activity, able to catalytic acetyl CoA to generate CoA.(5) The purified protein was used as antigen to immune rabbit to prepare polyclonal antibody successfully. iELISA analysis showed that the titer of the obtained antibody was1:128000. Western blot showed that the antibody could specifically combine with the MAT protein expressed in prokaryotic cell and HEK-293cell.In conclusion, we expressed and purified MAT protein. HPLC analysis of the in vitro enzymatic reaction products showed that the refolded protein had transferase activity. The polyclonal antibody with high affinity and specificity was generated successfully, which would provide a reliable foundation of studying the biological function of MAT in fatty acid metabolism.