Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris

Sarafotoxins are peptides isolated from the Atractaspisw snake venom. with strong constrictor effect on cardiac and smooth muscle. They are structural ly and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar st retches of sarafotoxin (SRTX) and spacer, in the present work, the recombin ant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pas toris. and secreted to the culture medium, Characterization by SDS-PAGE, im munoblot, mass spectrometry and biological activity, suggests that intact p recursor was expressed but processing into mature toxins also occurred. Fur thermore, our results indicate that the correct proportion of sarafotoxin t ypes 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 5 X 10(-10) M and 5 x 10(-11) M of sara fotoxin b, respectively. The enzymes responsible for the complete maturatio n of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation proces s. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs r elease. (C) 2001 Elsevier Science Ltd. All rights reserved.