Efficient expression and purification of recombinant sphignomyelinase D from Loxosceles laeta in lepidopteran larvaeas a candidate for antiserum production.

MC CALLUM GJ; ARREGUI MB; RUIZ M; MIRANDA MV; ARGNANI P; TARGOVNIK AM; CASCONE O; SMITH I; CALDERON L; FINGERMANN

Buenos Aires

Congreso; 20Th World Congress of the International Society on Toxinology. 8-13 septiembre 2019, Buenos Aires, Argentina.; 2019

Spiders from the genus Loxosceles are of great health importance for south america and thewhole world. Particularly in Argentina, Loxosceles laeta is the most abundant species. The bitesfrom Loxosceles laeta spiders can cause a wide array of toxic effects ranging fromdermonecrosis and complement-dependent haemolysis to disseminated vascular coagulationand renal failure. Current available treatment consists of antiserum produced in horses fromwhole venom obtained by electrostimulation of spiders which is a complicated process andproduces insufficient quantities.Sphingomyelinase D (Smase D) represents the main toxic component in Loxosceles laetavenom and as such constitutes and ideal candidate for recombinant expression for large scaleproduction of antisera. In this work we studied the expression and purification of recombinantSmase D (rSmase D) in baculovirus infected lepidopteran larvae as biofactories.Expression was successful in both Spodoptera frugiperda and Rachiplusia nu larvae, obtaining afinal yield after IMAC purification of approximately 2529 ± 48 and 1223 ± 120 mU/larvae foreach species respectively as measured by commercial colorimetric assay and with a high levelof purity. The resulting protein was not glycosylated and inoculation in rabbits demonstrateddermonecrotic lesions comparable to whole venom. Thus, Sphyngomyelinase D represents agood example of the use of insect larvae as a small bioreactor for the production ofrecombinant proteins in a cost-effective and easily scalable manner.