Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

• Published in: MicrobiologyOpen (Weinheim) Vol. 11; no. 3; pp. e1300 - n/a
• Main Authors: Cho, Ji Sung, Oh, Hye Ji, Jang, Young Eun, Kim, Hyun Jin, Kim, Areum, Song, Jong‐Am, Lee, Eun Jung, Lee, Jeewon
• Format: Journal Article
• Language: English
• Published: Bognor Regis John Wiley & Sons, Inc 01.06.2022; Wiley
• Subjects: Chemotherapy; Cloning; Design optimization; Gene expression; Genetic engineering; Glycine; Glycosylation; Granulocytes; human granulocyte colony‐stimulating factor; Leukocytes (granulocytic); Neutropenia; Optimization; Peptides; Protein folding; Proteins; Saccharomyces cerevisiae; Secretion; secretion efficiency; synthetic pro‐peptides; Vectors (Biology); Yeast; Yeasts
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.1300

Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony‐stimulating factor (hG‐CSF) in S. cerevisiae, a secretion‐enhancing peptide cassette including an hIL‐1β‐derived pro‐peptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters—N‐glycosylation, net negative charge balance, and glycine‐rich flexible linker—were investigated in batch cultures of S. cerevisiae. Using a three‐stage design involving screening, selection, and optimization, the production and secretion of hG‐CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG‐CSF produced by the optimized pro‐peptide after the final stage increased by 190% compared to that of the original pro‐peptide. Although hG‐CSF was used as the model protein in the current study, this strategy is applicable to the enhanced production of other heterologous proteins, using S. cerevisiae as the host. Saccharomyces cerevisiae is widely used for heterologous protein production but often secretes at comparatively low levels. To improve this, a secretion‐enhancing peptide cassette was added to the target protein and the pro‐peptide was optimized using three key parameters: N‐glycosylation, net negative charge balance, and glycine‐rich flexible linker. Production of the model protein, the human granulocyte colony‐stimulating factor, was increased by 190% compared to the original pro‐peptide. This stepwise rational design is a promising strategy applicable to other heterologous proteins.