Purification and partial characterization of Philibertain I, a cysteine protease from latex of Philibertia gilliesii hook

SEQUEIROS C, FERNÁNDEZ TAYELDÍN M, TORRES MJ, TREJO S, LÓPEZ LMI AND NATALUCCI C.

Bariloche

Congreso; The crude extract obtained fram latex of Philibertia gilliesii was; 2003

SAIB

The crude extract obtained fram latex of Philibertia gilliesii was partially purified by acetone fractionation as a first purification step that deprived the enzyme preparation ofmost soluble sugars, gums and low molecular weight peptides, retaining 73% of protein with 90% of proteolytic activity. Isoelectric focusing followed by zymogram analysis revealed the presence of severalbasic bands showing caseinolytic activity. According to pI values ofthe proteolytic components (8.75, 8.9, 9.8, and >10.25), the crude extract was purified by FPLC using a cation exchanger (SP-Sepharose Fast Flow equilibrated with 50 mM Tris-Gly buffer). To improve resolution, several saline gradients and different pH values were tested and the best resolution was obtained when a 0.1-0.4 M sodium chloride gradient at pH 9.0 was applied. The highest basic active fraction (philibertain 1)was homogeneous by IEF,SDS-PAGEand mass spectrometry (Mr 23530). partially purified by acetone fractionation as a first purification step that deprived the enzyme preparation ofmost soluble sugars, gums and low molecular weight peptides, retaining 73% of protein with 90% of proteolytic activity. Isoelectric focusing followed by zymogram analysis revealed the presence of severalbasic bands showing caseinolytic activity. According to pI values ofthe proteolytic components (8.75, 8.9, 9.8, and >10.25), the crude extract was purified by FPLC using a cation exchanger (SP-Sepharose Fast Flow equilibrated with 50 mM Tris-Gly buffer). To improve resolution, several saline gradients and different pH values were tested and the best resolution was obtained when a 0.1-0.4 M sodium chloride gradient at pH 9.0 was applied. The highest basic active fraction (philibertain 1)was homogeneous by IEF,SDS-PAGEand mass spectrometry (Mr 23530). Philibertia gilliesii was partially purified by acetone fractionation as a first purification step that deprived the enzyme preparation ofmost soluble sugars, gums and low molecular weight peptides, retaining 73% of protein with 90% of proteolytic activity. Isoelectric focusing followed by zymogram analysis revealed the presence of severalbasic bands showing caseinolytic activity. According to pI values ofthe proteolytic components (8.75, 8.9, 9.8, and >10.25), the crude extract was purified by FPLC using a cation exchanger (SP-Sepharose Fast Flow equilibrated with 50 mM Tris-Gly buffer). To improve resolution, several saline gradients and different pH values were tested and the best resolution was obtained when a 0.1-0.4 M sodium chloride gradient at pH 9.0 was applied. The highest basic active fraction (philibertain 1)was homogeneous by IEF,SDS-PAGEand mass spectrometry (Mr 23530). ICONICETfellow; 2CONICETResearcher Career;JCICResearcher Career. Worksupported by grants from ANPCyT, CONICET, CIC and UNLP.