title: Protein production in Yarrowia lipolytica via fusion to the secreted lipase Lip2p.
abstract: We established a strategy for protein production and purification via expression in Yarrowia lipolytica as Lip2p fusion protein. To evaluate the expression system a cysteine-rich miniprotein, an antibody fragment and an enzyme showing galactose oxidase activity were chosen. These proteins have varying disulfide bond content, size, and structural complexity. Endogenous lipase Lip2p was used as a fusion partner to direct the fused proteins to the extracellular medium. A linker sequence was introduced at the junction of Lip2p and the respective fused protein that contains a hexahistidine tag followed by a TEV protease cleavage site. This allows for a specific and simple purification via IMAC for capturing the secreted proteins from the supernatant followed by a second IMAC for removing all contaminants after proteolytic release of the protein of interest. Up to 174 mg/L fusion protein was obtained using shake flask cultivation. Functionality of each of the purified proteins was confirmed by individual assays. Expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for an online monitoring of accumulation of secreted fusion proteins in the medium by exploiting the intrinsic lipase activity of the fusion.
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstraße 22, 64287, Darmstadt, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hofmeyer
authors.forename: Siyavuya Ishmael
authors.initials: SI
authors.lastname: Bulani
authors.forename: Julius
authors.initials: J
authors.lastname: Grzeschik
authors.forename: Simon
authors.initials: S
authors.lastname: Krah
authors.forename: Bernhard
authors.initials: B
authors.lastname: Glotzbach
authors.forename: Christina
authors.initials: C
authors.lastname: Uth
authors.forename: Olga
authors.initials: O
authors.lastname: Avrutina
authors.forename: Michael
authors.initials: M
authors.lastname: Brecht
authors.forename: Hans Ulrich
authors.initials: HU
authors.lastname: Göringer
authors.forename: Petrus
authors.initials: P
authors.lastname: van Zyl
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase; D003545:Cysteine