Cloning, Expression, and Characterization of Protein Carboxyl O-methyltransferase from Porcine Brain

Protein carboxyl O-methyltransferase (E.C.2.1.1.24) may play a role in the repair of aged protein that is spontaneously incorporated with isoaspartyl residues. The porcine brain carboxyl O-methyltransferase was cloned in the pET32 rector, and overexpressed in E.coli (BL21) that harbors pETPCMT, which encodes 227 amino acids, including tagging proteins at the N-terminus. The protein sequence of the cloned porcine brain PCMT (r-pbPCMT) shares a 98% identity with that of human erythrocyte PCMT and rat brain PCMT. It is 100% identical with that of bovine brain. The r-pbPCMT was purified using Ni-NTA affinity chromatography and digested by enterokinase in order to remove the protein tags. Then Superdex 75HR gel filtration chromatography was performed. The r-pbPCMT exhibited similar in vitro substrate specificities with the PCMT that was purified from porcine brain. The molecular weight of the enzyme was estimated to be 24.5 kDa on the SDS polyacrylamide gel electrophoresis. The K_m value was 1.1 × 10^-7 M for S-adenosyl-L-methionine. S-adnosyl-L-homocysteine was a competitive type of inhibitor with the K_i value of 138 × 10^-4 M. The enzyme has optimal activity at pH 6.0 and 37°C. These results indicate that the expressed enzyme is functionally similar to the natural protein. It also suggests that it may be a suitable model to further understand the function of the mammalian enzyme.