Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 395, Issue 1, Pages 57-68Publisher
ELSEVIER SCIENCE INC
DOI: 10.1006/abbi.2001.2574
Keywords
cytochrome P4502B; N-terminal truncation; heterologous expression; membrane binding; oligomerization
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Funding
- NIEHS NIH HHS [ES06676, ES03619] Funding Source: Medline
- NIGMS NIH HHS [GM20674] Funding Source: Medline
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The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni2+-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and > 95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stercospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis. (C) 2001 Academic Press.
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