Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in <i>Saccharomyces cerevisiae</i>

Rakestraw, J. Andy; Sazinsky, Stephen L.; Piatesi, Andrea; Antipov, Eugene; Wittrup, K. Dane

doi:10.1002/bit.22338

Cited by 172 publications

(159 citation statements)

References 41 publications

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“…The V L and V H sequences of HyHEL-10 molecules were fused to the corresponding human IgG1 C L or C H 1, C H 2, and C H 3 sequences, respectively, generating light and heavy chains. Based on the literature, an evolved yeast MAT␣ pre-and propeptide sequence was selected to secrete IgG into the culture medium (25). All DNA sequences were codon optimized for expression in S. cerevisiae and assembled by DNA synthesis (Geneart, Germany).…”

Section: Figmentioning

confidence: 99%

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

Nasab

Aebi

Bernhard

et al. 2013

Appl Environ Microbiol

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bWe describe a novel synthetic N-glycosylation pathway to produce recombinant proteins carrying human-like N-glycans in Saccharomyces cerevisiae, at the same time addressing glycoform and glycosylation efficiency. The ⌬alg3 ⌬alg11 double mutant strain, in which the N-glycans are not matured to their native high-mannose structure, was used. In this mutant strain, lipidlinked Man 3 GlcNAc 2 is built up on the cytoplasmic side of the endoplasmic reticulum, flipped by an artificial flippase into the ER lumen, and then transferred with high efficiency to the nascent polypeptide by a protozoan oligosaccharyltransferase. Proteinbound Man 3 GlcNAc 2 serves directly as a substrate for Golgi apparatus-targeted human N-acetylglucosaminyltransferases I and II. Our results confirmed the presence of the complex human-like N-glycan structure GlcNAc 2 Man 3 GlcNAc 2 on the secreted monoclonal antibody HyHEL-10. However, due to the interference of Golgi apparatus-localized mannosyltransferases, heterogeneity of N-linked glycans was observed.

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Section: Figmentioning

confidence: 99%

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

Nasab

Aebi

Bernhard

et al. 2013

Appl Environ Microbiol

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“…When expressing a protein that has a secretory signal peptide, the protein is cotranslationally translocated into the endoplasmic reticulum (ER) lumen, where disulfide bond formation occurs with the initial glycosylation (9). The secretion of recombinant proteins is affected by their signal peptide sequences (10). A synthetic leader sequence increases human insulin precursor production, whereas the use of the ␣-factor leader sequence increased ␣-amylase production (7).…”

mentioning

confidence: 99%

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Bao

Huang

Petranović

et al. 2017

Appl Environ Microbiol

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The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous ␣-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased ␣-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous ␣-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-␣-glucosidase, indicating that this mechanism is of general relevance.IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future. KEYWORDS protein secretion, mitochondria, ERES, ROS, SEC16T he yeast Saccharomyces cerevisiae is a widely used cell factory for the production of recombinant proteins (1). The advantages of S. cerevisiae are fast growth, easy cultivation, and ability to perform posttranslational modifications and secrete proteins to the extracellular medium, which facilitates the purification of the protein (2, 3). However, S. cerevisiae naturally secretes only a few proteins, such as aspartyl protease, invertase, and ␣-factor pheromone (4, 5); therefore, it has evolved a secretory pathway with a relatively low capacity. Many different strategies have been used to engineer this pathway with the objective of improving heterologous protein production (6-8). When expressing a protein that has a secretory signal peptide, the protein is cotranslationally translocated into the endoplasmic reticulum (ER) lumen, where disulfide bond formation occurs with the initial glycosylation (9). The secretion of recombinant proteins is affected by their signal peptide sequences (10). A synthetic leader sequence increases human insulin precursor production, whereas the use of the ␣-factor leader sequence increased ␣-amylase production (7). Excessive mannose glycosylation of recombinant proteins can reduce the efficiency of protein secretion, and it was recently found that

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“…Yeast surface display is a well-established protein engineering technology that has been used for characterizing and screening protein-based inhibitors (40,(43)(44)(45) (Fig. S1B).…”

Section: Engineering the Kunitz Domain 2 (Kd2) Of Hai-1 To Bind Matrimentioning

confidence: 99%

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein

Mitchell

Kannan

Hunter

et al. 2018

Journal of Biological Chemistry

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Abstract:Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type 1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase binding and inhibition domain, while the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains, and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains 4 stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki =70 ± 5 pM, an increase of 120-fold compared to the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to wild-type KD1 but distinct from HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1. ________________________________________ High expression and dysregulated activity of the type II, membrane-anchored serine protease matriptase in the local tumor environment has been shown to correlate with poor patient prognosis in many human cancers including breast, colorectal, pancreatic, cervical, and prostate cancers (1-10). This dysregulation is partly driven by the high proteolytic processing and turnover of pro-hepatocyte growth factor (Pro-HGF) (9, 11) to a form of HGF that activates its cognate receptor, c-Met (12) (Fig. 1A). Matriptase is also known to activate other proteases and growth factors including urokinase plasminogen activator (uPA) (11), pro-macrophage stimulating protein (Pro-MSP) (13), and platelet derived growth factor-D (PDGF-D) (14), all of which play key roles in cancer growth and metastasis. Furthermore, matriptase has been identified as a critical driver of other diseases, including iron overload disease (15) and osteoarthritis (16), and has been shown to activate the human airway influenza virus (H1N1) (17) and human immunodeficiency virus (HIV) (18 (19)(20)(21)(22)(23). The activity of matriptase is regulated in healthy tissue by the serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1) (Fig. 1A). HAI-1 is primarily expressed on the surface of epithelial cells and naturally blocks the substrate-activating properties of matriptase (24)(25)(26), as well as other struc...

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Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in Saccharomyces cerevisiae

Cited by 172 publications

References 41 publications

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein

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