Nitrogen catabolite repressible GAP1 promoter, a new tool for efficient recombinant protein production in S. cerevisiae

Microbial Cell Factories

Research

Bruno André¹  Ahmad Merhi¹  Elsa Lauwers¹  Fabien Debailleul²  Cataldo Trubbia²  Jean-Marie Ruysschaert²  Cédric Govaerts²  Nancy Frederickx² 

[1] Lab Physiologie Moléculaire de la Cellule, Université Libre de Bruxelles, IBMM, rue des Pr. Jeener et Brachet 12, 6041, Gosselies, Belgium;S.F.M.B., Université Libre de Bruxelles, Blvd. du Triomphe, Bâtiment BC, local 1C4.208, B-1050, Bruxelles, Belgium;

关键词: Saccharomyces cerevisiae;    Protein expression;    Protein purification;    Heterologous expression;   

DOI  :  10.1186/1475-2859-12-129

 received in 2013-05-30, accepted in 2013-12-18,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundDecades of work requiring heterologous expression of eukaryotic proteins have shown that no expression system can be considered as the panacea and the appropriate expression strategy is often protein-dependent. In a large number of cases, yeasts have proven to be reliable organisms for heterologous protein expression by combining eukaryotic cellular organization with the ease of use of simpler microorganisms.ResultsDuring this work, a novel promoter system based on the nitrogen catabolite regulation has been developed to produce the general amino acid permease (Gap1) in its natural host, the yeast Saccharomyces cerevisiae. A simple purification protocol was also established that allows to purify milligrams of Gap1 from cells cultivated in a five liters bio-reactor. In order to test the ability of the system to be used for expression of other proteins, the yeast specific transporter of γ-aminobutyric acid (Uga4), a human vesicular transporter of glutamate (Vglut1) and a small secreted glycoprotein (MD-2) were also expressed using the nitrogen catabolite regulation. All proteins were fused to GFP and their presence and localization were confirmed by western blot analysis and fluorescence microscopy.ConclusionsOur work shows that the nitrogen catabolite repressible GAP1 promoter can be used to obtain high levels of recombinant protein while allowing for large biomass production in S. cerevisiae. This approach can be used to express membrane and soluble proteins from higher eukaryotes (from yeast to human). Therefore, this system stands as a promising alternative to commonly used expression procedure in yeasts.

【 授权许可】

Unknown   
© Debailleul et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.