Expression, purification, and characterization of human malonyl-CoA decarboxylase

• Published in: Protein expression and purification 2004-04, Vol.34 (2), p.261-269
• Main 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
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Carboxy-Lyases - genetics; Carboxy-Lyases - isolation & purification; Carboxy-Lyases - metabolism; Cloning, Molecular; Escherichia coli - genetics; Expression and purification; Human malonyl-CoA decarboxylase; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Homology; Substrate Specificity
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2003.11.023

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 K m value of approximately 330–520 μM and a turnover rate ( k cat) of 13–28 s −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.