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 constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. 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 kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.
Chatani
E
E
Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Tanimizu
N
N
Ueno
H
H
Hayashi
R
R
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D002417:Cattle; D004795:Enzyme Stability; D006031:Glycosylation; D013058:Mass Spectrometry; D010179:Pancreas / Q000201:enzymology; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D002241:Carbohydrates; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic