The Cloning And Expression Of Oligomycin Sensitivity-Conferring Protein (OSCP) Gene In E.coli

The cloning and expression of Oligomycin Sensitivity Conferring Protein (OSCP) gene in E.coliIt was reported that oligomycin sensitivity-conferring protein (OSCP), a subunit of proton F0Fl-ATPase/ATP synthase in the mitochondrial inner membranes, as a new estradial binding protein. This finding suggestes that mitochondrial ATPase/ATP synthase could be a potential target for estradiol or compounds with similar structures. Moreover, OSCP has been isolated and purified from the neuronal membrane of rat brain, it is about a 23 KDa protein. But the clone and expression of OSCP in vitro is not reported. Here, we cloned the OSCP of the female rat and expressed it in E.coli, moreover, through SDS-PAGE and western-blotting we testify the truth of the expressed OSCP in order to study the structure and function as a membrane receptor for estrogen. Objective To clone and express the oligomycin sensitivity-conferring protein (OSCP) gene of rat in E.coli. Methods Extract the whole RNA from the rat brain tissue, and then a couple primers were designed for PCR according to the known sequence of OSCP gene. The OSCP gene obtain -ed by PCR technique was cloned into plasmid pGEM-T easy vector directio -nally, the constructed recombinant plasmid pGEM-T easy-OSCP was trans- ferred into E.coli JM 109. The transformatants were screened and identified by restriction analysis. Thecloned genes were sequenced by Sanger's method. Modify the two clones of OSCP gene by restrict endonuclease and recombine them to obtain the correct sequence, and then clone it into a prokaryotic expre -ssion vector pET-28c (+) to construct a recombinant plasmid PET-28c (+)-OSCP; The E.coli cells were transformed with the recombinant plasmid and the transformants were induced to express the recombinant protein at 37 and was identified. Results The size of amplified OSCP gene was 700bp. The correct recombinant plasmid pGEM-T easy-OSCP was isolated and confirmed by restriction analysis. DNA sequencing showed the DNA sequence of cloned gene was almost the same as the published sequence. Digestion with restrict endonuclease and sequencing show that the modification of/for the sense mutation of OSCP is success. Digestion with restrict endonuclease also shows the prokaryotic expression vector pET-28c (+)-OSCP was constructed. The expressing product by 1PTG was detected by SDS-PAGE and western blotting, the result shows an expression band about 23 KD(a) was found and the expressing product has immunogenicity. Conclusion The OSCP was successfully expressed and the expressing product has immunogenicity. The expression of OSCP in E.coli provided the basic material for the study of structure and function of OSCP.