HIGH-LEVEL EXPRESSION OF RECOMBINANT APLYSIA ADP-RIBOSYL CYCLASE IN PICHIA-PASTORIS BY FERMENTATION

Cyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD(+), is rapidly emerging as an endogenous modulator of Ca2+-i nduced Ca2+ release mechanisms in various cellular systems. ADP ribosy l cyclase, first isolated from the marine invertebrate Aplysia califor nica, cyclizes NAD(+) to cADPR. In this study we have utilized the met hylotrophic yeast Pichia pastoris to express high levels of this enzym e. The cyclase construct consisted of the soluble domain, with isoleuc ine (25 residues following the initial methionine) as the N-terminus, cloned in frame with the yeast ct-factor mating signal sequence. Cycla se yeast transformants were screened using the Zeocin (phleomycin from Streptomyces verticillus) selectable marker which resulted in 100% ac tive transformation. All active clones comprised the methanol utilizat ion slow (Mut(s)) phenotype. The protein was expressed using the tight ly regulated methanol-inducible alcohol oxidase (AOX1) promoter and th e Saccharomyces cerevisiae alpha-factor mating secretion signal. Using high biomass fermentations, up to 300 mg/liter of cyclase was achieve d. SDS-PAGE analysis revealed that the heterologous protein comprised nearly 90-95% of the total protein secreted extracellularly. The enzym e characteristics of the recombinant cyclase compared favorably with t hose of the native enzyme. The yeast expression system can thus produc e gram quantities of this novel protein. (C) 1997 Academic Press.