Construction of recombinant NNMT plasmid and expression in E. coli

Objective To prepare Nicotinamide N-methyltransferase (NNMT) antigen with fusion protein of GST by pGEX-4T-2 expression system. Methods According to the nucleotide bank of NCBI (gi:12652950),DNA primers with BamHI/XhoI site were designed. Total RNA was isolated from the human liver tissue using TRIzol reagent and was reverse transcripted into the c DNA. The polymerase chain reaction (PCR) was used to amplify the NNMT sequence with its cDNA as template and primers designed on the basis of the NNMT DNA sequence. The PCR product was ligated into the vector pGEX-4T-2 after T-A clone and subclone. The recombinnat plasmid was transferred into E.coli BL-21-STAR(DE3) and the GST-NNMT fusion protein was expressed by inducing with IPTG. The fusion protein was purified by glutathione sepharose 4B affinity chromatography. DNA sequencing, SDS-PAGE and Western-blot were used to verify the plasmid and the fusion protein. Results Restriction analysis and DNA sequencing confirmed the correct sequence and insertion site of the recombinant pGEX-4T-2/NNMT. Expressed fusion protein was mainly in soluble supernatant and accounted for about 20% of the total bacterial proteins. SDS-PAGE and Western-blot results indicated that the fusion protein was GST-NNMT. Conclusion The expression system of pGEX-4T-2/NNMT was successfully constructed.