Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
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
Autore:
GUZOV VM; ZELKO IN; CHUDAEV MV; GUZOVA JA; CHUNG BC; USANOV SA;
Indirizzi:
BYELARUSSIAN ACAD SCI,INST BIOORGAN CHEM,UL ZHODINSKAYA 5-2 MINSK 220141 BYELARUS BYELARUSSIAN ACAD SCI,INST BIOORGAN CHEM MINSK 220141 BYELARUS ACAD SINICA,INST MOL BIOL TAIPEI 11529 TAIWAN
Titolo Testata:
Biochemistry
fascicolo: 10, volume: 61, anno: 1996,
pagine: 1242 - 1252
SICI:
0006-2979(1996)61:10<1242:EOFAHC>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
HIGH-LEVEL EXPRESSION; YEAST SACCHAROMYCES-CEREVISIAE; INDUCIBLE EXPRESSION; ADRENAL-CORTEX; ENZYME; INSERTION; SYSTEM; SIGNAL; CELLS; CDNA;
Keywords:
CYTOCHROME P-450-C21; EXPRESSION IN ESCHERICHIA COLI; METAL CHELATE AFFINITY CHROMATOGRAPHY; PURIFICATION; STEROIDS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
45
Recensione:
Indirizzi per estratti:
Citazione:
V.M. Guzov et al., "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", Biochemistry, 61(10), 1996, pp. 1242-1252

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 recombinantcytochrome 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 membranes. Modification of the N- and C-terminal sequences of cytochromeP-450c21 does not change the K-m and V-max for progesterone and 17 alpha-hydroxyprogesterone hydroxylation. The recombinant cytochrome P-450c21 expressed in E, coli was purified from solubilized bacterial membranes using metal chelate affinity chromatography. The highly purifiedhemoprotein migrates in SDS-PAGE as a single band corresponding to molecular weight 54 kD and shows type I binding spectra with progesterone and 17 alpha-hydroxyprogesterone. Hydroxylation activity of the purified cytochrome P-450c21 was reconstituted in the presence of purifiedNADPH-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-hydroxyprogesterone and progesterone, and the V-max 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 mu M for 17 alpha-hydroxyprogesterone and 14.7 mu M for progesterone.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 22:49:44