Expression, purification, and characterization of human malonyl-CoA decarboxylase |
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Authors: | Zhou Demin Yuen Phoebe Chu Donald Thon Vicki McConnell Steve Brown Steven Tsang Andria Pena Michael Russell Anna Cheng Jie-Fei Nadzan Alex M Barbosa Miguel S Dyck Jason R B Lopaschuk Gary D Yang Guang |
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Affiliation: | Department of Discovery Biology, Chugai Pharma USA, LLC, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA. |
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Abstract: | The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1). For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD. |
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Keywords: | Author Keywords: Human malonyl-CoA decarboxylase Expression and purification |
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