Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications.
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.
Mast
Natalia
N
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031, USA.
Andersson
Ulla
U
Nakayama
Kazuo
K
Bjorkhem
Ingemar
I
Pikuleva
Irina A
IA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000072556:Cholesterol 24-Hydroxylase; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D013250:Steroid Hydroxylases; D000072556:Cholesterol 24-Hydroxylase