Multicopy overexpression of bovine pancreatic trypsin inhibitor saturates the protein folding and secretory capacity of Saccharomyces cerevisiae.
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.
Parekh
R
R
Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
Forrester
K
K
Wittrup
D
D
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D007611:Aprotinin