Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast... 10.1002/bit.260420113.abs Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid‐k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid‐k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFα1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80–90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long‐term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid‐containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid‐k locus was observed. © 1993 John Wiley & Sons, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biotechnology and Bioengineering Wiley

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Download PDF
8 pages

Loading next page...
 
/lp/wiley/improved-protein-synthesis-and-secretion-through-medium-enrichment-in-kR05zxkF7v

References (35)

Publisher
Wiley
Copyright
Copyright © 1993 John Wiley & Sons, Inc.
ISSN
0006-3592
eISSN
1097-0290
DOI
10.1002/bit.260420113
pmid
18609652
Publisher site
See Article on Publisher Site

Abstract

10.1002/bit.260420113.abs Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid‐k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid‐k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFα1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80–90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long‐term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid‐containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid‐k locus was observed. © 1993 John Wiley & Sons, Inc.

Journal

Biotechnology and Bioengineering – Wiley

Published: Jun 5, 1993

There are no references for this article.