Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications

Abstract

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3–27), and, in addition, Δ46A1H had a 4× His-tag fused to the C-terminus; HΔ46A1 had the N-terminal 4× His-tag; HΔ46A1Δ had a 4× His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494–499); and Δ46A1Δ lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390–650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, Δ46A1Δ and HΔ46A1Δ, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3–0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.