Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was const ructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 prom oter was used for efficient secretion. Approximately 5 mg of soluble enzyme s were secreted per liter of the culture, but one half of them were glycosy lated. After a series of purifications by cation-exchange chromatography, t he glycosylated enzyme was removed and the pure recombinant soluble unglyco sylated RNase A was obtained in the final yield of 1 mg per liter of the cu lture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k(cat), 60-70% of the activit y of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety s eemed tb destabilize the enzyme differently from RNase B since T-m of the g lycosylated RNase A was decreased by 6 degreesC. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc, The N34A mutant RNase A, in w hich the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-l inked.