oa Cloning, overexpression and molecular characterization of human mast cell carboxypeptidase A

Background: Mast cell proteases represent major protein components of secretory granules. The proteases are classified into a chymases, tryptases and carboxypeptidase (hMC-CPA). Little is known about the function of human lung carboxypeptidase A (hMC-CP) except for its ability to cleave angiotensin I. This suggests that the human mast cell carboxypeptidase A may play a role in the hypertension disease. Objectives: 1) Making human lung cDNA library and isolating the human lung carboxypeptidase A; 2) Cloning and expressing the full length PCR products into the overexpression vector pET28a and into pYES2; 3) Molecular characterization of the recombinant proteins. Methods: Human lung cDNA was prepared and PCR was carried out using DNA primers of the carboxypeptidase A (hMC-CPA).The isolated gene fragment was subcloned into the overexpression vector pET28a and transformed into E. coli DE3 for expression. The gene was also subcloned into the pYES2 vector for expression in yeast. The expression in E. coli has been validated by Western Blot analysis using four different antibodies. The purification was carried out using Ni²+ column chromatography. The recombinant protein was further characterized using MS and peptide amino acid sequencing. Results: We cloned and overexpressed the gene in E.coli using the His-tag vector pET28a. The protein expression in E. coli has been validated by Western blot analysis using four different carboxypeptidase A antibodies. The protein however, has been expressed in the form of inclusion bodies. Despite the fact that we could obtain soluble CPA at lower IPTG concentration and lower induction temperature we decide to subclone and express the gene in Saccharomyces cerevisiae. We successfully managed to subclone the gene in pYES2 vector and to produce a soluble form of the CPA in Saccharomyces cerevisiae. The hrMC-CPA expressed in Saccharomyces cerevisiae in frame with six His-tags. The protein therefore was purified by one step purification using Ni column. Conclusion: The cloning and overexpression of the CPA in soluble form will pave the way for molecular characterization studies of this enzyme and to shed more light on its biological function.