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.
Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
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Parekh
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Forrester
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Wittrup
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