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 journalArticle

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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

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Affinity chromatography
Cytochromes
Escherichia coli
Purification
Metals
17-alpha-Hydroxyprogesterone
Progesterone
Complementary DNA
Membranes
NADPH-Ferrihemoprotein Reductase
Hydroxylation
Spectrophotometry
Titration
NADP
Recombinant Proteins
Histidine
Liver
Enzymes

All Science Journal Classification (ASJC) codes

  • Chemistry(all)

Cite this

Guzov, V. M., Zel'ko, I. N., Chudaev, M. V., Guzova, Y. A., Chung, B. C., & Usanov, S. A. (1996). Expression of active human cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and one-step purification by metal-affinity chromatography. Biokhimiya, 61(10), 1758-1771.

Expression of active human cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and one-step purification by metal-affinity chromatography. / Guzov, V. M.; Zel'ko, I. N.; Chudaev, M. V.; Guzova, Yu A.; Chung, Bon Chu; Usanov, S. A.

In: Biokhimiya, Vol. 61, No. 10, 01.10.1996, p. 1758-1771.

Research output: Contribution to journalArticle

Guzov, VM, Zel'ko, IN, Chudaev, MV, Guzova, YA, Chung, BC & Usanov, SA 1996, 'Expression of active human cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and one-step purification by metal-affinity chromatography', Biokhimiya, vol. 61, no. 10, pp. 1758-1771.
Guzov, V. M. ; Zel'ko, I. N. ; Chudaev, M. V. ; Guzova, Yu A. ; Chung, Bon Chu ; Usanov, S. A. / Expression of active human cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and one-step purification by metal-affinity chromatography. In: Biokhimiya. 1996 ; Vol. 61, No. 10. pp. 1758-1771.
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