PURIFICATION OF AN ASPARTIC PROTEINASE FROM SOLANUM ELAEAGNIFOLIUM FRUITS (SEAP-1

TORRES NÁGERA, MA; FERNANDO F. MUÑOZ,; LOPEZ- LOPOZ, LI; DE LA CRUZ GALICIA, G; SONIA YESENIA SILVA BELMARES; MARÍA G. GUEVARA.

Congreso; Reunion Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Solanum elaeagnifolium is an endemic plant from the northeast of Mexico. This plant in some places of Mexico has been used for decades in the manufacture of artisanal filata-type asadero cheese. The milk-clotting activity of S. elaeagnifolium has been attributed to aspartic proteinases. The aim of this work was to purify aspartic proteinases from fruits of S. elaeagnifolium. Purification was performed from lyophilized fruits of S. eleagnifolium by ammonium sulfate precipitation; ion exchange chromatography and Pepstatin A affinity chromatography. SDS- PAGE analysis of proteins eluted from the affinity column shown only one protein band corresponding to 59 kDa., approximately. Identity of this protein band as aspartic proteinase was demonstrated by Western blot analysis, using as primary antibody IgG-anti StAP1 (Solanum tuberosum aspartic proteinase 1). Results obtained from gel filtration chromatography (Superose 12) demonstrates that, isolated SeAP1 is a monomeric enzyme, with an estimated molecular weight of 52 kDa., approximately. Additionally, we determined that, like almost monomeric plant APs, SeAP-1 exerts cytotoxic activity towards plant pathogens in a dose- dependent manner. These results suggest the presence of saposin- like domain into the sequence of the mature SeAP-1 and therefore, new biotechnological applications for SeAP-1.