Optimization of amino acid supplements for heterologous protein secretion in Saccharomyces cerevisiae

1994 ◽  
Vol 8 (3) ◽  
pp. 161-166 ◽  
Author(s):  
K. D. Wittrup ◽  
V. Benig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Protein Secretion ◽  
Heterologous Protein ◽  
Heterologous Protein Secretion

scholarly journals Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

2000 ◽  
Vol 66 (11) ◽  
pp. 4940-4944 ◽  
Author(s):  
C. M. J. Sagt ◽  
B. Kleizen ◽  
R. Verwaal ◽  
M. D. M. de Jong ◽  
W. H. Müller ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Secretion ◽  
Heterologous Protein ◽  
Consensus Sequence ◽  
Glycosylation Site ◽  
Antibody Fragments ◽  
Terminal Region ◽  
Economic Feasibility ◽  
Heterologous Proteins ◽  
Heterologous Protein Secretion

ABSTRACT Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama VHH antibody fragments by S. cerevisiae orPichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

scholarly journals An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lisa Ann Burdette ◽  
Han Teng Wong ◽  
Danielle Tullman-Ercek
Keyword(s):  
Protein Secretion ◽  
Salt Concentration ◽  
Type Iii Secretion ◽  
Heterologous Protein ◽  
Carbon Sources ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Heterologous Protein Secretion ◽  
Extracellular Environment

Abstract Background Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer. Results We dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements. Conclusions We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

Heterologous protein secretion by Candida utilis

2013 ◽  
Vol 97 (16) ◽  
pp. 7357-7368 ◽  
Author(s):  
Maya Kunigo ◽  
Christoph Buerth ◽  
Denis Tielker ◽  
Joachim F. Ernst
Keyword(s):  
Protein Secretion ◽  
Candida Utilis ◽  
Heterologous Protein ◽  
Heterologous Protein Secretion
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