Expression of Carcinoscorpius rotundicauda Factor C in Pichia pastoris

Abstract

Factor C from the amebocytes of the estuarine horseshoe crab Carcinoscorpius rotundicauda (CrFC) is a 132-kDa glycosylated protein. CrFC cDNA has been cloned and expressed in the methylotrophic yeast Pichia pastoris. Multiple copies of the cDNA were integrated into the yeast genome. Methanol-induced expression in shake flask cultures generated high levels of longer than full-length transcript, indicating that P. pastoris did not recognize the termination signals present in CrFC, cDNA, leading to transcript elongation by approximately 800 nucleotides. CrFC mRNA was found to be present maximally at the fifth hour of induction. The recombinant FC protein (rFC) of the predicted molecular mass was translated in maximal amounts at an unusually early time point of eight hours after induction. A truncated portion of CrFC comprising the endotoxin binding region was also expressed using the same system. Neither the native signal sequence nor the Saccharomyces cerevisiae α mating factor was capable of directing secretion of this mosaic protein. However, the Pichia-derived intracellular recombinant protein was immunoreactive and possibly membrane bound.