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

V. M. Gdzov, I. N. Zelko, M. V. Chudaev, J. A. Guzova, B. C. Chung, S. A. Usanov

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Abstract

Functionally active human cytochrome P-450c21 has been expressed in Escherichia coli and purified to apparent homogeneity. To increase the expression level, the N-terminal sequence of the cDNA was modified. The C-terminal sequence of the cDNA was modified by inserting an additional four histidine residues to allow the one-step enzyme purification procedure using metal chelate affinity chromatography. The recombinant cytochrome P-450c21 is expressed in E. coli in the amount 40-50 nmoles per liter of the growth medium and is inserted into the bacterial membranes. Modification of the N- and C-terminal sequences of cytochrome P-450c21 does not change the Km and Vmax for progesterone and 17α-hydroxyprogesterone hydroxylation. The recombinant cytochrome P-450c21 expressed in E. coli was purified from solubilized bacterial membranes using metal chelate affinity chromatography. The highly purified hemoprotein migrates in SDS-PAGE as a single band corresponding to molecular weight 54 kD and shows type I binding spectra with progesterone and 17α-hydroxyprogesterone. Hydroxylation activity of the purified cytochrome P-450c21 was reconstituted in the presence of purified NADPH-cytochrome P-450 reductase and an NADPH-regenerating system. The Km values for the highly purified recombinant cytochrome P-450c21 in the reconstituted system were 12.2 μM and 3.21 μM for 17α-hydroxyprogesterone and progesterone, and the Vmax values were 192.9 nmoles/min and 198 nmoles/min per nmole P-450c21, respectively. The dissociation constant determined from the difference binding spectra was 31.1 μM for 17α-hydroxyprogesterone and 14.7 μM for progesterone.

Original languageEnglish
Pages (from-to)1242-1252
Number of pages11
JournalBiochemistry (Moscow)
Volume61
Issue number10
Publication statusPublished - Dec 1 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry

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