AIM:To clone and express the human colon mast cellcarboxypeptidase(MC-CP)gene.METHODS:Total RNA was extracted from colon tissue,andthe cDNA encoding human colon mast cell carboxypeptidasewas amplified by reverse-transcription PCR(RT-PCR).Theproduct cDNA was subcloned into the prokaryotic expressionvector pMAL-c2x and eukaryotic expression vector pPIC9K toconstruct prokaryotic expression vector pMAL/human MC-CP(hMC-CP)and eukaryotic pPIC9K/hMC-CP.The recombinantfusion protein expressed in E.coli was induced with IPTG andpurified by amylose affinity chromatography.After digestionwith factor Xa,recombinant hMC-CP was purified by heparinagarose chromatography.The recombinant hMC-CP expressedin Pichia pastoris(P.pastoris)was induced with methanol andanalyzed by SDS-PAGE,Western blot,N-terminal amino acidsequencing and enzyme assay.RESULTS:The cDNA encoding the human colon mast cellcarboxypeptidase was cloned,which had five nucleotidevariations compared with skin MC-CP cDNA.The recombinanthMC-CP protein expressed in E.coliwas purified with amyloseaffinity chromatography and heparin agarose chromatography.SDS-PAGE and Western blot analysis showed that therecombinant protein expressed by E,coli had a molecularweight of 36 kDa and reacted to the anti-native hMC-CPmonoclonal antibody(CA5).The N-terminal amino acidsequence confirmed further the product was hMC-CP.E,coligenerated hMC-CP showed a very low level of enzymaticactivity,but P.pastoris produced hMC-CP had a relativelyhigh enzymatic activity towards a synthetic substratehippuryl-L-phenylalanine.CONCLUSION:The cDNA encoding human colon mast cellcarboxypeptidase can be successfully cloned and expressedin E.coli and P.pastoris,which will contribute greatly to thefunctional study on hMC-CP. AIM: To clone and express the human colon mast cellcarboxypeptidase (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) was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K toconstruct 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 andpurified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparinagarose chromatography. The recombinant hMC-CP was expressed with Pichia pastoris (P. pastoris) was induced with methanol andalyzed by SDS- PAGE, Western blot, N-terminal amino Results of acidsequencing and enzyme assay .RESULTS: The cDNA encoding the human colon mast cellcarboxypeptidase was cloned, which had five nucleotidevariations compared with skin MC-CP cDNA.The recombinanthM C-CP protein expressed in E. coli was purified with amyloseaffinity chromatography and heparin agarose chromatography. SDS-PAGE and Western blot analysis showed that therecombinant protein expressed by E, coli had a molecular weight of 36 kDa andacted to the anti-native hMC-CPmonoclonal antibody (CA5). The N-terminal amino acid sequence further further demonstrated that the product was hMC-CP. E. coligenerated hMC-CP showed a very low level of enzymaticactivity, but P. pastoris produced hMC-CP had a relativelyhigh enzymatic activity towards a synthetic substratehippuryl-L-phenylalanine.CONCLUSION: The cDNA encoding human colon mast cellcarboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the functional study on hMC-CP.