Isolation, Expression, and Purification of Recombinant Crotamine in Comparison with Native Crotamine

Background: In Southern Pacific rattlesnakes, (Crotalus oreganus helleri), a total of seven isoforms of crotamine (CRO) were identified through a cDNA library constructed from the venom gland of a Crotalus o.h. housed in the Serpentarium (Coh534). Crotamine has been suggested to have analgesic activity, which could be beneficial to the pharmaceutical industry in the treatment of chronic pain and various muscular disorders. Methods: Crude venom from the offspring, Coh792, of Coh534 was fractionated using High Performance Liquid Chromatography (HPLC) and yielded a positively charged molecule in fraction one. This basic polypeptide belongs to a group of myotoxins that have natural cell penetrating peptides, and is approximately 42 amino acids long and weighs 7kDa. In this experiment we are isolating, expressing, and purifying recombinant crotamine (r-CRO) using Talon Metal Affinity Resin, and comparing its effects against native crotamine from Coh792 using Cytotoxicity Assays. Results: r-CRO was successfully expressed in E. Coli BL21 Cells, and was purified using Affinity Chromatography. Upon injection r-CRO proved to have neurotoxic and myotoxic effects of temporary hind leg paralysis in female BALB/c mice. Based on the Cytotox96 Assays performed, CRO and r-CRO proved to be cytotoxic on C2C12 murine myoblasts. Conclusion: While the mechanism of action is still unknown, it is believed that CRO acts on a voltage-gated ion channel. Studying these recombinant proteins will provide important information regarding their activities, mechanism of action, and how they relate to their sequences. These studies will also provide a limitless supply of information that can be used in research and therapeutically as a safe alternative to narcotics in the treatment of chronic pain and various muscular disorders.