Multicopy overexpression of bovine pancreatic trypsin inhibitor saturates the protein folding and secretory capacity of Saccharomyces cerevisiae

Protein Expr Purif. 1995 Aug;6(4):537-45. doi: 10.1006/prep.1995.1071.

Abstract

Bovine pancreatic trypsin inhibitor (BPTI) was expressed and secreted from a synthetic gene as a model system for the study of protein folding and secretion in Saccharomyces cerevisiae. The efficiency of different leader sequences in directing BPTI secretion was examined, and up to 11 micrograms/ml of active BPTI was secreted. In some fusion constructs, inefficient proteolytic processing by Kex2p, Ste13p, and signal peptidase were observed immediately adjacent to the BPTI N terminus. Insertion of dipeptide spacers improved endoproteolytic processing substantially but the level of secretion was unchanged. Overexpression from a 2-microns multicopy vector results in essentially unchanged BPTI secretion as compared to expression from a single copy centromere vector. BPTI expressed from a multicopy vector accumulates intracellularly in an unfolded form, indicating that available secretory chaperones and foldases can be saturated by increasing the rate of BPTI synthesis.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aprotinin / biosynthesis
  • Aprotinin / chemistry
  • Aprotinin / genetics*
  • Base Sequence
  • Cattle
  • DNA Primers / genetics
  • Endoplasmic Reticulum / metabolism
  • Gene Amplification
  • Gene Expression
  • Genes, Synthetic
  • Genetic Vectors
  • Molecular Sequence Data
  • Protein Folding
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Trypsin Inhibitors / biosynthesis
  • Trypsin Inhibitors / chemistry
  • Trypsin Inhibitors / genetics*

Substances

  • DNA Primers
  • Recombinant Fusion Proteins
  • Trypsin Inhibitors
  • Aprotinin