Production of recombinant rat hepatic histidase.
Rat liver histidase was expressed in E. coli by using a PCR product of the coding sequence obtained from the rat liver cDNA of histidase cloned in the expression vector pRSET. The construct (pRSET-HAL) produced a fusion protein containing a tail of polyhistidine. The expression product was purified with a resin containing Ni+ that retains proteins with polyhistidine fragments. pRSET-HAL was analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence. Native rat liver histidase was also purified and it had a Mr of 72 kDa. An antiserum against native histidase was obtained in rabbit. Western blot analysis revealed one band of 72 kDa observed in membranes containing purified histidase or rat liver high speed supernatants. The same antiserum also detected in cell lysates of E. coli transformed with the pRSET-HAL plasmid a single band of 74 kDa of the recombinant histidase before cleavage with enterokinase. After the proteolysis, the Western blot analysis showed a single band of approximately 72 kDa. Kinetic analysis of the recombinant histidase showed similar Km and Vmax compared with native histidase.
Ortiz
Victor
V
Depto. Fisiologia de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrici6n Salvador Zubirán, México, DF.
Torres
Nimbe
N
Tovar
Armando R
AR
D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D006638:Histidine Ammonia-Lyase / Q000138:chemical synthesis* / Q000302:isolation & purification*; D008099:Liver / Q000737:chemistry* / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar
D006638:Histidine Ammonia-Lyase