title: Improved expression of single-chain antibodies in Ustilago maydis.
abstract: To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.
authors.affiliation: Heinrich Heine University Düsseldorf, Institute for Microbiology, Bldg. 26.12.01, 40204 Düsseldorf, Germany.
authors.forename: Parveen
authors.initials: P
authors.lastname: Sarkari
authors.affiliation: Heinrich Heine University Düsseldorf, Institute for Microbiology, Bldg. 26.12.01, 40204 Düsseldorf, Germany.
authors.forename: Michèle
authors.initials: M
authors.lastname: Reindl
authors.affiliation: Heinrich Heine University Düsseldorf, Institute for Microbiology, Bldg. 26.12.01, 40204 Düsseldorf, Germany.
authors.forename: Janpeter
authors.initials: J
authors.lastname: Stock
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Straße 10, 35043 Marburg, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Müller
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Straße 10, 35043 Marburg, Germany.
authors.forename: Regine
authors.initials: R
authors.lastname: Kahmann
authors.affiliation: Heinrich Heine University Düsseldorf, Institute for Microbiology, Bldg. 26.12.01, 40204 Düsseldorf, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Feldbrügge
authors.affiliation: Heinrich Heine University Düsseldorf, Institute for Microbiology, Bldg. 26.12.01, 40204 Düsseldorf, Germany. Electronic address: kerstin.schipper@uni-duesseldorf.de.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Schipper
mesh_terms: D002688:Chitinases / Q000096:biosynthesis* / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis*; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014588:Ustilago / Q000235:genetics
chemical_list: D000939:Epitopes; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; C121990:CTS1 protein, S cerevisiae; D002688:Chitinases
keywords: Protease; Protein expression; Single-chain antibody; Unconventional secretion; Ustilago maydis