(188ap) Expression, Purification, and Characterization of the New Recombinant Crotamine Isoform from the Venom Gland of Crotalus Oreganus Helleri on Antimicrobial Activity

Crotamine, a highly basic, non-enzymatic toxin, is the major toxic component found in the venom of the South American rattlesnake, Crotalus durissus terrificus. Based on the three-dimensional structure of crotamine, its cysteine-pairing pattern and folding structure is very similar to those of the other small toxins identified in different rattlesnake venoms and the antimicrobial peptide human β-defensins. Native, recombinant, and synthesized crotamine (YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG) has been found to contain antimicrobial activity against a wide variety of bacteria and fungi. In the present work, we aim to express recombinant propeptide (start codon methionine) and mature (start codon tyrosine) crotamine-like isoforms, helleramines, cloned from the venom gland cDNA library of Crotalus oreganus helleri and to characterize their antimicrobial activity against Gram-negative and Gram-positive bacteria. Mature helleramines 6O01 cloned into pGEX-4T-1 vector (6O01-pGEX-4T-1), were expressed in Escherichia coli BL21. The N-terminus Glutathione S-transferase (GST)-tagged fusion protein was purified using Glutathione Sepharose 4B resins. Purified tag-cleaved recombinant helleramines will be further investigated for their antimicrobial activities. The production of recombinant helleramines with biological activity may provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development.