Expression of active human cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and one-step purification by metal-affinity chromatography

V. M. Guzov, I. N. Zel'ko, M. V. Chudaev, Yu A. Guzova, Bon Chu Chung, S. A. Usanov

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17α-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17α-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 μM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17α-hydroprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17α-hydroxyprogesterone were 14.7 and 31.1 μM, respectively.

Original languageEnglish
Pages (from-to)1758-1771
Number of pages14
JournalBiokhimiya
Volume61
Issue number10
Publication statusPublished - Oct 1 1996

All Science Journal Classification (ASJC) codes

  • Chemistry(all)

Fingerprint Dive into the research topics of 'Expression of active human cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and one-step purification by metal-affinity chromatography'. Together they form a unique fingerprint.

Cite this