title: Expression and secretion of a CB4-1 scFv-GFP fusion protein by fission yeast.
abstract: There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion.
authors.affiliation: PomBioTech GmbH, Campus Building A1-1, Saarbrücken, Germany.
authors.forename: Julia Maria
authors.initials: JM
authors.lastname: Naumann
authors.forename: Gabriele
authors.initials: G
authors.lastname: Küttner
authors.forename: Matthias
authors.initials: M
authors.lastname: Bureik
mesh_terms: D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins