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

Authors
 Koh Eun-Jin  ;  Shim Ki-Shuk  ;  Kim Hyun-Kyu  ;  Park Ki-Moon  ;  Lee Suk-Chan  ;  Kim Jung-Dong  ;  Yoo Sun-Dong  ;  Chi Sang-Chul  ;  Hong Sung-Youl 
Citation
 JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY , Vol.34(6) : 559-565, 2001 
Journal Title
 JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY 
ISSN
 1225-8687 
Issue Date
2001
Keywords
Cloning ; Expression ; Porcine brain protein carboxyl O-methyltransferase
Abstract
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 vector, 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 polyacrylamidegel electrophoresis. The Km value was 1.1 ×10-7M for S-adenosyl-L-methionine. S-adnosyl-L-homocysteine was a competitive type of inhibitor with the Ki value of 1.38 ×10-4M. The enzyme has optimal activity at pH 6.0 and 37℃. 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.
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Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Biochemistry and Molecular Biology (생화학-분자생물학교실) > 1. Journal Papers
Yonsei Authors
Koh, Eun Jin(고은진) ORCID logo https://orcid.org/0000-0001-8967-6266
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/143229
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