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Abstract
Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed
as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris.
By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5mg Hsp70-gloshedobin
per liter of culture was achieved by methanol induction. The fusion protein secreted
in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose
FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa
according to SDS-PAGE analysis, and exhibited fibrinogenolytic activity that preferentially
degraded fibrinogen alpha-chain. The enzyme also degraded fibrinogen beta-chain to
a lesser extent, while showing no degradation toward the gamma-chain. A fibrinogen
clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range
reported for other thrombin-like enzymes. Hsp70-gloshedobin had strong esterase activity
toward the chromogenic substrate N alpha-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this
activity was optimal at pH 7.5 and 50 degrees C, and was completely inhibited by PMSF,
but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical
properties of gloshedobin. Although applying a fusion partner with very big molecular
weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin
without affecting its fibrinogenolytic activity. And this positive result may provide
an alternative strategy for the expression of thrombin-like enzymes in microbial system.