| Total RNA was extracted from the venom gland of the snake Gloydius shedaoensis. The cDNA of gloshedobin, a thrombin-like enzyme from Gloydius shedaoensis, was cloned and amplified by RT-PCR. Assay of the nucleotide sequence of the cDNA allowed postulation of the complete amino acid sequence for gloshedobin. Its amino acid sequence exhibits significant homology with those of other thrombin-like snake venom enzymes. Based on the homology, the catalytic residues and disulfide bridges of gloshedobin were deduced to be as follows : His43, Asp88, Ser182 and Asp176; and disulfide bridges, Cys7-141, Cys 28-44, Cys 76-234, Cys120-188, Cys152-167 and Cys178-213. Although most of snake thrombin-like enzymes are known to have two sites for glycosylation (Asn-X-Thr), fibrinolysin has only one possible glycosylation site. Asn124-Ser125-Thr126. Similarly, the full length nucleotide sequence of the cDNA of gussurobin, a thrombin-like enzyme from Gloydius ussuriensis. was obtained. Compared with gloshedobin, gussurobin has three different amino acid residues: Gln66 vs. Glu66; Gly117 vs. Asp117; Thr 225 vs. He225. Although gussurobin showed 98% homology on the nucleotide sequence with gloshedobin. its enzymatic activity showed less similarity with gloshedobin. Therefore, these three residues' replacement may influence the stability of the advanced structure of the enzyme.Both the mature genes of gloshedobin and gussurobin were cloned into thevector pET-32a(+), strain BL21(DE3), to study their expression in prokaryotic cell. The gene was expressed under T7 promoter with a fusion protein partner of Thx.Tag and a 6x His.Tag at its N-terminal. Having been induced by IPTG for 4 hours, the recombinant enzyme was examined in the cytoplasm by SDS-PAGE analysis.The mature gene of gloshedobin was inserted into the expression vector pPIC9K. The yeast Pichia pastoris strain GS115 was applied to study the expression. The transfermants with highest resistance to G418 (4 g/l in YPD) were screened to study their expression level in 150 ml shaking flask. Having been induced with methanol for 36 hours, the target enzyme could be examined in the supernatant by measuring amidolytic activity. Compared with minimal medium, rich medium leads to higher expression level up to 4 mg target protein/L . The optimal culture conditions we-e obtained as follow5 : complex medium buffered to pH 6.0; harvesting cells after 36 hours of methanol induction.The recombinant gloshedobin in the supernatant was separated and purified by followed two combinations of chromatographic methods. One is a combination of ion exchange chromatography on Q Sepharose FF, hydrophobic interaction chromatography on Phenyl HP, gel filtration chromatography on Sephadex 200 and higher resolution ion exchange chromatography on Mono Q. The other is a combination of ion exchange chromatography on Q Sepharose FF, two affinity chromatography on Con A Sepharose 4B and Benzamidine Sepharose 6BCL sequentially. According tothe purity and the activity recovery, the recombinant enzyme was well purified by both of these two separation combinations. Like natural gloshedobin, the recombinant enzyme exhibited strong esterase activity using tripeptide p-nitroanilide derivatives as substrate, but hydrolyzed N a -p-tosyl-L-arginine methyl ester (TAME) or N a -benzoyl-L-arginine ethyl ester ( BAEE ) very weakly. This recombinant protein displayed extreme instability at 37? in neutral buffer but higher stability at 0?. pH is not a key factor to affect its stability while the optimal pH for its enzymatic activity is pH 8.0. The recombinant gloshedobin binding to the affinity adsorbent Con A indicated that it is a glycoprotein. The molecular mass of this enzyme is about 28 kDa according to SDS-PAGE electrophoresis analysis. Compared with the theoretical molecular mass of 25902.5 Da calculated by adding up all amino acids, the difference of 2 kDa probably contributed to the sugar chain. This means the mass percentage of the sugar moietv is about 7%.
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