¢ñ Type Arginase Gene Cloning And Expression

Arginase, which converts arginine to ornithine and urea, is a kind of enzyme in urea cycle. It plays a significant role in N mechabolism. As a semi-necessary amino acid, arginine is only necessary for fast-grown tissue, especially for the tumor tissue. So arginase can serve as an anticarcinogenic agent by depriving of arginine, which does much more harm to cancer cell than to normal tissue. The objective of this paper is to clone and express human arginase I gene (hA I) with molecular cloning technique.The hA I was amplified from human liver RNA by RT-PCR, followed by a routine sequencing assay to identify its nucleic acid sequence. Then the sequence was analyzed and compared with the data from GeneBank. The result demonstrated that hA I was amplified correctly.The hA I was inserted into pPIC9K vector containing AOX1 promoter and α-factor signal sequence to construct a recombinant plasmid pPIC9K-hA I. Then pPIC9K-hA I was transformed into Pichia pastoris SMD1168 to construct recombinant Pichia pastoris strain SMD1168 (pPIC9K- hA I ). The recombinant strain was induced by 0.5% methanol. After being induced for 120h, no enzyme was detected in supernatant by either enzyme activity assay or SDS-PAGE. But the cell lysate showed enzyme activity of 0.372U per gram cells (wet weight) .The hA I was also inserted into pET30a vector containing T7 promoter to construct a recombinant plasmid pET30a-hA I. Then pET30a-hA I was transformed into E. coli BL21(DE3) to construct recombinant E coli strain BL21(DE3)( pET30a-hA I ). The recombinant strain was induced by 1mM IPTG. With the induction of 1mM IPTG for 7h, the BL21(DE3)(pET30a-hA I ) showed enzyme activity of 34.6U per gram cells (wet weight) . SDS-PAGE showed that the molecular weight of the protein expressed was about 35kD, which was accordant with the theoretical value.