MOLECULAR ENGINEERING OF MICROSOMAL P450 2A-4 TO A STABLE, WATER-SOLUBLE ENZYME

Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mou se microsomal P450 2a-4, was genetically engineered and expressed in E scherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic pep tide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738), The expression level of Pe pti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any dete rgents during the chromatographic steps. The purified Pepti-P450 retai ned the spectral and catalytic properties of the unmodified enzyme wit h a similar K-m value for steroid 15 alpha-hydroxylase activity (19.7 mu M in comparison to 14.2 mu M of the wildtype). Gel permeation chrom atography showed that the purified Pepti-P450 in the detergent-free bu ffer was an oligomer with an approximate molecular mass of 450 kDa. Th e replacement of the hydrophobic anchor domain with an amphipathic hel ix such as peptitergent, therefore, may provide a general method for e ngineering membrane-bound P450s to soluble enzymes. (C) 1995 Academic Press, Inc.