Efficient expression and purification of recombinant sphingomyelinase D from Loxosceles laeta in Lepidopteran larvae as a candidate for antiserum production

GREGORIO MC CALLUM; PATRICIA ARGNANI; IGNASIO SMITH; MARIANA ARREGUI; ALEXANDRA M. TARGOVNIK; LEANDRO PEDRO CALDERÓN; MARCELO RUÍZ; OSVALDO CASCONE; MATÍAS FINGERMANN; MARÍA VICTORIA MIRANDA

Buenos Aires

Congreso; 20Th World Congress of the International Society on Toxinology; 2019

20Th World Congress of the International Society on Toxinology

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. Thebites from Loxosceles laeta spiders can cause a wide array of toxic effects ranging fromdermonecrosis and complement-dependent haemolysis to disseminated vascularcoagulation and renal failure. Current available treatment consists of antiserum produced inhorses from whole venom obtained by electrostimulation of spiders which is a complicatedprocess and produces 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 largescale production of antisera. In this work we studied the expression and purification ofrecombinant Smase D (rSmase D) in baculovirus infected lepidopteran larvae asbiofactories.Expression was successful in both Spodoptera frugiperda and Rachiplusia nu larvae,obtaining a final yield after IMAC purification of approximately 2529 ± 48 and 1223 ± 120mU/larvae for each species respectively as measured by commercial colorimetric assay andwith a high level of purity. The resulting protein was not glycosylated and inoculation inrabbits demonstrated dermonecrotic lesions comparable to whole venom. Thus,Sphyngomyelinase D represents a good example of the use of insect larvae as a smallbioreactor for the production of recombinant proteins in a cost-effective and easily scalablemanner