Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant thaumatin in yeast.
When the heterologous proteins thaumatin and bovine prochymosin are produced in yeast cells as a fusion with the yeast invertase secretory signal peptide, less than 2% of the product is secreted in a biologically active form into the medium. The remainder accumulates intracellularly in a misfolded conformation. We investigated whether this poor secretion can be improved by overexpression of binding protein (BiP) one of the major chaperones in eukaryotic cells. Indeed, a tenfold increase in the level of binding protein, as a result of the introduction of extra copies of the kar2 gene into yeast cells containing a single, integrated copy of the invertase/prochymosin fusion gene, caused more than a 20-fold increase in the amount of extracellular prochymosin. By additional disruption of the PMR1 gene of these cells we were able to obtain secretion of virtually all of the prochymosin produced. Export of thaumatin, on the other hand, was not significantly stimulated by binding protein overexpression.
Harmsen
M M
MM
Department of Biochemistry and Molecular Biology, IMBW, BioCentrum Amsterdam, Vrije Universiteit, The Netherlands.
Bruyne
M I
MI
Raué
H A
HA
Maat
J
J
D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000737:chemistry / Q000378:metabolism*; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013549:Sweetening Agents; D043324:beta-Fructofuranosidase
D004792:Enzyme Precursors; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013549:Sweetening Agents; C003427:thaumatin protein, plant; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; C032443:prorennin; D012085:Chymosin