title: Production of the rat complement regulator, Crry, as an active soluble protein in Pichia pastoris.
abstract: In this report, we describe the use of the methylotrophic yeast Pichia pastoris for the production of the rat complement regulator, Crry. Crry normally exists as an intrinsic membrane protein containing six to seven short consensus repeats (SCRs), a transmembrane region, and a cytoplasmic tail. To produce Crry as a soluble recombinant protein, nucleotides encoding the five N-terminal SCRs from the rat Crry cDNA were amplified by PCR, and cloned into the P. pastoris expression vector, pPIC9. This vector contains the yeast alpha-factor signal sequence, thereby leading to secretion of recombinant protein. This construct was subsequently integrated into P. pastoris strain GS115 genomic DNA. Secreted soluble Crry was produced by induction of the AOX1 promoter with methanol. Recombinant Crry protein was purified to homogeneity by sequential Mono Q and Mono P chromatography. The protein was highly active toward the alternative and classical pathways of complement, inhibiting the latter by approximately 90% at a concentration of 15 nM. The P. pastoris system offers an efficient method for the production of soluble recombinant Crry. Production of active rat Crry offers opportunities to study long-term models of disease in rats, which has not been possible with available heterologous complement inhibitors.
authors.affiliation: Department of Medicine, University of Chicago, Illinois 60637, USA.
authors.forename: C
authors.initials: C
authors.lastname: He
authors.forename: J J
authors.initials: JJ
authors.lastname: Alexander
authors.forename: A
authors.initials: A
authors.lastname: Lim
authors.forename: R J
authors.initials: RJ
authors.lastname: Quigg
mesh_terms: D000818:Animals; D000954:Antigens, Surface; D004274:DNA, Recombinant; D005822:Genetic Vectors; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics / Q000502:physiology; D010843:Pichia / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D051381:Rats; D011956:Receptors, Cell Surface; D011951:Receptors, Complement / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012995:Solubility; D014170:Transformation, Genetic
chemical_list: D000954:Antigens, Surface; C513105:Cr1l protein, rat; D004274:DNA, Recombinant; D010455:Peptides; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor