Use of phosphopeptide to purify industrially important proteins starting from the venom of Crotalus durisuss terrificus USE OF PHOSPHOPEPTIDE TO PURIFY INDUSTRIALLY IMPORTANT PROTEINS STARTING FROM THE VENOM OF Crotalus durisuss terrificus

S. L. SAAVEDRA; G. ACOSTA; L. ÁVILA; S. L. GIUDICESSI; S. A. CAMPERI; F. ALBERICIO; J. C. DOKMETJIAN; O. CASCONE; M. C. MARTÍNEZ CERON

CABA

Congreso; World Congress of the International Society on Toxinology; 2019

The International Society on Toxinology

Crotalus durissus terrificus (Cdt) main toxic compound of its venom is crotoxin (almost 50% of dry venom), is a β-neurotoxin composed by two subunits non-covalently bonded, one inactive and the other a phospholipase A2. Crotamine (Ctm) is nonenzymatic (about 12% of the dry weight of the crude venom of crotamine-positive Cdt) and both have interesting pharmacological uses, as antiviral, antibacterial and against tumor cells. Peptides have proved to be very useful ligands for the purification of numerous molecules such as antibodies, toxins, among other proteins. The aim of this work was to design a phosphopeptide (P-Lys) with a phosphoserine in its sequence. Its synthesis was carried out in solid phase on Rink-Amide-ChemMatrix resin. Then, it was immobilized in NHS-agarose to be used as an affinity chromatographic matrix and process conditions were evaluated using SDS-PAGE 15% and SDS-PAGE-tricine gels, and protein concentration and enzymatic activity were evaluated. For the chromatography, 100 μl of the venom (150 mg/ml) of a 1/20 dilution in the adsorption buffer was applied into the matrix. In the best conditions, the percentage of adsorption reached 70% and when performing the gels one band was observed in eluate 1 which correspond to the MW of Ctm (5 kDa) and three bands were observed in eluate 2, of which one corresponds to the MW of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry were identified. Only eluate 2 presented enzymatic activity. The yield of the process exceeded 100% (it has been described that free PLA2 has greater activity than complexed)and with a purity of 90% for PLA2. For Ctm the purity was 76%. Through the design of a phosphopeptide it was possible to recover both, PLA2 and Ctm with an adequate purity from a sample of Cdt venom in a single chromatographic step.