Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

Swennen, Dominique; Paul, Marie-Françoise; Vernis, Laurence; Beckerich, Jean-Marie; Fournier, Alain; Gaillardin, Claude

doi:10.1099/00221287-148-1-41

Cited by 47 publications

(28 citation statements)

References 50 publications

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“…The pGAA plasmid containing the Arxula adeninivorans glucoamylase (GAA) coding sequence under the control of the hp4d hybrid promoter (Swennen et al, 2002) was used to transform wild-type and mutant haploid strains. To express GAA, the Y. lipolytica cells were cultivated on rich YPD in 50 mM phosphate buffer, pH 6?8, at 28 uC.…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

2004

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In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae α-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of the Y. lipolytica proteins, including the previously isolated YlMnn9p, in the N-glycosylation pathway, a phenotypic analysis of the disrupted strains ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9 and ΔYlmnn9ΔYloch1 was performed. Disruption of the YlMNN9, YlANL1 and YlOCH1 genes caused an increased sensitivity to SDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YlMnn9p and YlAnl1p in the N-glycosylation process. These mutant strains, ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9 and ΔYlmnn9ΔYloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.

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Section: Methodsmentioning

confidence: 99%

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

2004

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“…46 Notably, K. lactis is also able to produce single-chain Fv antibodies. 47,48 New concepts in recombinant yeast vaccine technology are the use of inactivated whole yeast cells, 49 or antigen display at the yeast cell surface. 50,51 These technologies are of great interest for the development of oral vaccines in veterinary medicine using both conventional and nonconventional yeast strains.…”

Section: -39mentioning

confidence: 99%

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

Legastelois

Buffin

Peubez

et al. 2016

Human Vaccines & Immunotherapeutics

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The increasing demand for recombinant vaccine antigens or immunotherapeutic molecules calls into question the universality of current protein expression systems. Vaccine production can require relatively low amounts of expressed materials, but represents an extremely diverse category consisting of different target antigens with marked structural differences. In contrast, monoclonal antibodies, by definition share key molecular characteristics and require a production system capable of very large outputs, which drives the quest for highly efficient and cost-effective systems. In discussing expression systems, the primary assumption is that a universal production platform for vaccines and immunotherapeutics will unlikely exist. This review provides an overview of the evolution of traditional expression systems, including mammalian cells, yeast and E.coli, but also alternative systems such as other bacteria than E. coli, transgenic animals, insect cells, plants and microalgae, Tetrahymena thermophila, Leishmania tarentolae, filamentous fungi, cell free systems, and the incorporation of non-natural amino acids.

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“…The yeast K. lactis is one of the model systems utilized for heterologous protein production in the food and pharmacology industries (18,19,35,39,44,51). In this work, we reported the characterization of a non-glucose-repressible mutant of K. lactis to be used as a host for recombinant protein production.…”

Section: Discussionmentioning

confidence: 99%

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

Donnini

Farina

Neglia

et al. 2004

Appl Environ Microbiol

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The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1␤ compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position ؉527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N-and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.Yeasts are very useful hosts for the production of heterologous proteins. The yeast Kluyveromyces lactis presents several advantages over other yeast species. It is positive for lactose fermentation, is able to grow on cheap substrates such as residual whey from dairy industries, and has competitive secretory properties, excellent large-scale fermentation characteristics, and food grade status; also, both episomal and integrative expression vectors are available for it (for reviews, see references 20, 40, and 50). Its ability to secrete heterologous proteins into the medium at a concentration higher than that secreted by Saccharomyces cerevisiae was demonstrated previously (50), although the secretory and glycosylation processes and their regulation are still poorly understood for K. lactis (1,42,43).For K. lactis, the regulation of primary carbon metabolism differs markedly from that for S. cerevisiae and reflects the dominance of respiration over fermentation that is typical for the majority of yeast species (7). In K. lactis, respiration is not repressed by glucose, and fermentative and oxidative metabolism can take place simultaneously. Glucose repression, however, does exist: several enzymes that are required for alternate carbohydrate metabolism have been shown to be subject to glucose repression (6,13,17,25,30). The K. lactis genes involved in glucose repression include RAG1, encoding a lowaffinity glucose permease (23, 48); DGR151 (or RAG5), encoding the single hexokinase of this yea...

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Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

Cited by 47 publications

References 50 publications

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

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