Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications |
| |
Authors: | Mast Natalia Andersson Ulla Nakayama Kazuo Bjorkhem Ingemar Pikuleva Irina A |
| |
Institution: | Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031, USA. |
| |
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, Delta 46A1H had a 4x His-tag fused to the C-terminus; H Delta 46A1 had the N-terminal 4x His-tag; H Delta 46A1 Delta had a 4x His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and Delta 46A1 Delta 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, Delta 46A1 Delta and H Delta 46A1 Delta, 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. |
| |
Keywords: | P450 CYP46A1 N-terminal truncation C-terminal truncation Membrane localization Aggregation Proline-rich region |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|