Production of the Rat Complement Regulator, Crry, as an Active Soluble Protein inPichia pastoris

Abstract

In this report, we describe the use of the methylotrophic yeastPichia pastorisfor 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 theP. pastorisexpression vector, pPIC9. This vector contains the yeast α-factor signal sequence, thereby leading to secretion of recombinant protein. This construct was subsequently integrated intoP. pastorisstrain GS115 genomic DNA. Secreted soluble Crry was produced by induction of theAOX1promoter 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 ≈90% at a concentration of 15 nm. TheP. pastorissystem 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.