Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris.
Sarafotoxins are peptides isolated from the Atractaspis snake venom, with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer. In the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris, and secreted to the culture medium. Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5x10(-10)M and 5x10(-11)M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release.
Borgheresi
R A
RA
Lab. Farmacologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo, Brazil. robarbor@usp.br
Palma
M S
MS
Ducancel
F
F
Camargo
A C
AC
Carmona
E
E
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D010843:Pichia / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis* / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D014757:Viper Venoms / Q000096:biosynthesis* / Q000494:pharmacology
D011498:Protein Precursors; D011994:Recombinant Proteins; D014757:Viper Venoms; C057354:sarafotoxins s6