Expression and characterization of Flavikunin: A Kunitz-type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps.

Trancriptomic analysis of the venom gland cDNA library of Bungarus flaviceps revealed Kunitz-type serine protease inhibitor as one of the major venom protein families with three groups A, B, C. One of the group B isoforms named Flavikunin, which lacked an extra cysteine residue involved in disulfide bond formation in ß-bungarotoxin, was synthesized, cloned, and overexpressed in Escherichia coli. To decipher the structure-function relationship, the P1 residue of Flavikunin, histidine, was mutated to alanine and arginine. Purified wild-type and mutant Flavikunins were screened against serine proteases-thrombin, factor Xa, trypsin, chymotrypsin, plasmin, and elastase. The wild-type and mutant Flavikunin (H∆R) inhibited plasmin with an IC 50 of 0.48 and 0.35 µM, respectively. The in-silico study showed that P1 residue of wild-type and mutant (H∆R) Flavikunin interacted with S1' and S1 site of plasmin, respectively. Thus, histidine at the P1 position was found to be involved in plasmin inhibition with mild anticoagulant activity.