12] Purification of recombinant soluble forms of furin produced in chinese hamster ovary cells

Abstract

Publisher Summary This chapter describes the purification of recombinant soluble forms of furin produced in Chinese hamster ovary (CHO) cells. It also discusses transfection experiments using, human colon carcinoma LoVo cells as host. To produce large amounts of a secretory form of furin, the complementary DNA (cDNA) for a furin protein truncated up to the transmembrane domain from the COOH terminus is expressed under the control of the simian virus 40 (SV40) early promoter in CHO cells. The expression of furin in CHO cells is then amplified using methotrexate. Assay for furin is based on the coexpression of furin and a substrate precursor in cultured cells, in which furin can cleave various precursor proteins after sequences fitting the RXK/RR motif. Furin is also capable of cleaving short fluorogenic peptides with a sequence fitting the RXK/RR motif. All purification steps are performed at 0–4°, and all buffers used contain 1 mM CaCl2. Furin is extremely unstable at low protein concentrations; the final preparations which are stored at –80° lose activity over time.