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

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 330–520μM 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.