Cloning and expression of human colon mast cell carboxypeptidase

AIM:To clone and express the human colon mast cell carboxypeptidase (MC-CP) gene.METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR).The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to construct prokaryotic expression vector pMAL/human MC-CP(hMC-CP) and eukaryotic pPIC9K/hMC-CR The recombinant fusion protein expressed in Eco/iwas induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoHs(Rpastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid sequencing and enzyme assay.RESULTS:The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.co/iwas purified with amylose affinity chromatography and heparin agarose chromatography.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecula rweight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E.coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.CONCLUSION: The cDNA encoding human colon mast cell carboxypeptidase can be successfully cloned and expressed in E.coliand P. pastoris, which will contribute greatly to the functional study on hMC-CP.

AIM:To clone and express the human colon mast cell carboxypeptidase(MC-CP)gene. METHODS:Total RNA was extracted from colon tissue,and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR(RT-PCR).The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to construct prokaryotic expression vector pMAL/human MC-CP (hMC-CP)and eukaryotic pPIC9K/hMC-CP.The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography.After digestion with factor Xa,recombinant hMC-CP was purified by heparin agarose chromatography.The recombinant hMC-CP expressed in Pichia pastoris(P.pastoris)was induced with methanol and analyzed by SDS-PAGE,Western blot,N-terminal amino acid sequencing and enzyme assay. RESULTS:The cDNA encoding the human colon mast cell carboxypeptidase was cloned,which had five nucleotide variations compared with skin MC-CP cDNA.The recombinant hMC-CP protein expressed in E.coliwas purified with amylose affinity chromatography and heparin agarose chromatography. SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E,coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody(CA5).The N-terminal amino acid sequence confirmed further the product was hMC-CP.E,coli generated hMC-CP showed a very low level of enzymatic activity,but P.pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine. CONCLUSION:The cDNA encoding human colon mast cell carboxypeptidase can be successfully cloned and expressed in E.coli and P.pastoris,which will contribute greatly to the functional study on hMC-CP.