TU Darmstadt / ULB / TUbiblio

Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p.

Hofmeyer, Thomas and Bulani, Siyavuya Ishmael and Grzeschik, Julius and Krah, Simon and Glotzbach, Bernhard and Uth, Christina and Avrutina, Olga and Brecht, Michael and Göringer, H. Ulrich and van Zyl, Petrus and Kolmar, Harald (2014):
Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p.
In: Molecular biotechnology, pp. 79-80, 56, (1), ISSN 1559-0305, [Article]

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.

Item Type: Article
Erschienen: 2014
Creators: Hofmeyer, Thomas and Bulani, Siyavuya Ishmael and Grzeschik, Julius and Krah, Simon and Glotzbach, Bernhard and Uth, Christina and Avrutina, Olga and Brecht, Michael and Göringer, H. Ulrich and van Zyl, Petrus and Kolmar, Harald
Title: Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p.
Language: English
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.

Journal or Publication Title: Molecular biotechnology
Volume: 56
Number: 1
Divisions: 10 Department of Biology
10 Department of Biology > Postranscriptional Gene Regulation and RNA Therapeutics
Date Deposited: 23 Jul 2013 11:15
Export:

Optionen (nur für Redakteure)

View Item View Item