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

Functionally active human cytochrome P-450c21 has been expressed in Es cherichia coli and purified to apparent homogeneity. To increase the e xpression level, the N-terminal sequence of the cDNA was modified. The C-terminal sequence of the cDNA was modified by inserting an addition al four histidine residues to allow the one-step enzyme purification p rocedure using metal chelate affinity chromatography. The recombinant cytochrome P-450c21 is expressed in E. coil in the amount 40-50 nmoles per liter of the growth medium and is inserted into the bacterial mem branes. Modification of the N- and C-terminal sequences of cytochrome P-450c21 does not change the K-m and V-max for progesterone and 17 alp ha-hydroxyprogesterone hydroxylation. The recombinant cytochrome P-450 c21 expressed in E, coli was purified from solubilized bacterial membr anes using metal chelate affinity chromatography. The highly purified hemoprotein migrates in SDS-PAGE as a single band corresponding to mol ecular weight 54 kD and shows type I binding spectra with progesterone and 17 alpha-hydroxyprogesterone. Hydroxylation activity of the purif ied cytochrome P-450c21 was reconstituted in the presence of purified NADPH-cytochrome P-450 reductase and an NADPH-regenerating system. The K-m values for the highly purified recombinant cytochrome P-450c21 in the reconstituted system were 12.2 mu M and 3.21 mu M for 17 alpha-hy droxyprogesterone and progesterone, and the V-max values were 192.9 nm oles/min and 198 nmoles/min per nmole P-450c21, respectively. The diss ociation constant determined from the difference binding spectra was 3 1.1 mu M for 17 alpha-hydroxyprogesterone and 14.7 mu M for progestero ne.