title: High-level expression of recombinant Aplysia ADP-ribosyl cyclase in offhia pastoris by fermentation.
abstract: Cyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD+, is rapidly emerging as an endogenous modulator of Ca2(+)-induced Ca2+ release mechanisms in various cellular systems. ADP ribosyl cyclase, first isolated from the marine invertebrate Aplysia californica, cyclizes NAD+ to cADPR. In this study we have utilized the methylotrophic yeast Pichia pastoris to express high levels of this enzyme. The cyclase construct consisted of the soluble domain, with isoleucine (25 residues following the initial methionine) as the N-terminus, cloned in frame with the yeast alpha-factor mating signal sequence. Cyclase yeast transformants were screened using the Zeocin (phleomycin from Streptomyces verticillus) selectable marker which resulted in 100% active transformation. All active clones comprised the methanol utilization slow (Muts) phenotype. The protein was expressed using the tightly regulated methanol-inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae alpha-factor mating secretion signal. Using high biomass fermentations, up to 300 mg/liter of cyclase was achieved. SDS-PAGE analysis revealed that the heterologous protein comprised nearly 90-95% of the total protein secreted extracellularly. The enzyme characteristics of the recombinant cyclase compared favorably with those of the native enzyme. The yeast expression system can thus produce gram quantities of this novel protein.
authors.affiliation: Department of Physiology, University of Minnesota, Minneapolis 55455, USA.
authors.forename: C
authors.initials: C
authors.lastname: Munshi
authors.forename: H C
authors.initials: HC
authors.lastname: Lee
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001048:Aplysia / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D005285:Fermentation; D005470:Fluorometry; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009244:NAD+ Nucleosidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D036541:ADP-ribosyl Cyclase; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1