Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group
Section snippets
General chemicals and reagents
The following chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO): IPTG, δ-ALA, thiamine, imidazole hydrochloride, DTT, sodium cholate, PMSF, leupeptin, aprotinin, bestatin, TPCK, NADPH, Coomassie brilliant blue R, DLPC, TFA, lauric acid, and 12-hydroxylauric acid. Additional hydroxylauric acid metabolites were synthesized as previously described [22]. BSTFA was purchased from Supelco Inc. (Bellefonte, PA). Restriction enzymes and other DNA-modifying enzymes were from New
Construct selection
PCR was used to generate CYP4B1 genes possessing a variety of 5′ structural modifications that would result in the predicted amino acid sequences shown in Table 1. The unmodified, wild-type cDNA was amplified and cloned, and named CYP4B1-native. The CYP4B1#1 construct had the first 8 amino acid residues replaced by the Barnes sequence, MALLLAVF, a procedure that has been widely adopted and proven to be successful for the expression in E. coli of certain other eukaryotic P450s [23], [28], [29],
Conclusions
Bacterial expression of rabbit CYP4B1 requires structural modification of the N-terminus. These modifications differentially affect cellular toxicity and the degree to which the enzyme is processed post-translationally by E. coli. Of considerable interest is the observation that the bacterially expressed CYP4B1 holoenzyme contains >98.5% covalently bound heme prosthetic group. E. coli-expressed CYP4B1 retains the catalytic activity and regioselectivity of laurate hydroxylation, described
Acknowledgements
This study was supported by NIH Grant GM43511 (AER). BRB was supported by NIH Predoctoral Training Grant GM07750. MJC wishes to thank Belinda J. Cheesman for kind support. The authors would also like to thank Dr. R.W. Estabrook for providing pCWhum3A4HT.
References (40)
- et al.
Methods Enzymol.
(2002) - et al.
J. Biol. Chem.
(2000) - et al.
Biochem. Biophys. Res. Commun.
(1998) - et al.
Toxicol. Appl. Pharmacol.
(1993) - et al.
Biochem. Pharmacol.
(1997) - et al.
Biochem. Pharmacol.
(1995) - et al.
J. Biol. Chem.
(2001) - et al.
J. Biol. Chem.
(2002) - et al.
Arch. Biochem. Biophys.
(2000) - et al.
Arch. Biochem. Biophys.
(1997)