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

Archives of Biochemistry and Biophysics
Natalia MastIrina A Pikuleva

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 ...Continue Reading

Associated Clinical Trials

Oct 16, 2018·Irina Pikuleva, PhD

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Citations

Feb 21, 2006·Applied Microbiology and Biotechnology·Raku ShinkyoToshiyuki Sakaki
May 6, 2008·Acta Crystallographica. Section D, Biological Crystallography·Mark Andrew WhiteIrina A Pikuleva
Mar 10, 2010·Biochemistry. Biokhimii︠a︡·L A NovikovaV M Shkumatov
Jun 6, 2009·Annual Review of Biochemistry·David W RussellTiina Kotti
Jul 16, 2008·Proceedings of the National Academy of Sciences of the United States of America·Natalia MastIrina A Pikuleva
Mar 15, 2011·Expert Opinion on Drug Metabolism & Toxicology·Dmitri R Davydov
Jun 16, 2009·Protein Expression and Purification·Thomas HamannBirger Lindberg Møller
Mar 28, 2007·Journal of Biotechnology·Norbert W KolarPieter Swart
Dec 20, 2013·The Journal of Biological Chemistry·Natalia MastIrina A Pikuleva
Jan 26, 2006·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Irina A Pikuleva
Jun 24, 2017·The Journal of Biological Chemistry·Natalia MastIrina A Pikuleva
Apr 9, 2016·The Journal of Biological Chemistry·Kyle W AndersonIrina A Pikuleva
Dec 11, 2007·Chemical Research in Toxicology·Elizabeth M J Gillam
Jun 6, 2021·The Journal of Steroid Biochemistry and Molecular Biology·Natalia PutkaradzeRita Bernhardt
Mar 2, 2005·Biochemistry·Natalia MastIrina A Pikuleva

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