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

Biosci Biotechnol Biochem. 2000 Nov;64(11):2437-44. doi: 10.1271/bbb.64.2437.

Abstract

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.

MeSH terms

  • Animals
  • Carbohydrates / chemistry
  • Cattle
  • Enzyme Stability
  • Glycosylation
  • Mass Spectrometry
  • Pancreas / enzymology
  • Pichia / genetics*
  • Plasmids / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Ribonuclease, Pancreatic / biosynthesis*
  • Ribonuclease, Pancreatic / chemistry
  • Ribonuclease, Pancreatic / genetics
  • Ribonuclease, Pancreatic / isolation & purification
  • Solubility

Substances

  • Carbohydrates
  • Recombinant Proteins
  • Ribonuclease, Pancreatic