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

Fabien Debailleul,Cataldo Trubbia,Elsa Lauwers,Bruno André,Ahmad Merhi,Jean-Marie Ruysschaert,Cédric Govaerts,Nancy Frederickx

doi:10.1186/1475-2859-12-129

Fabien Debailleul, Cataldo Trubbia + Show 6 more

Open Access

https://doi.org/10.1186/1475-2859-12-129

Copy DOI

Journal: Microbial Cell Factories	Publication Date: Dec 1, 2013
Citations: 51	License type: cc-by

Affiliation: Université Libre de Bruxelles

Abstract

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.

Highlights

Decades 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
Membrane proteins destined to the plasma membrane traffic through the endoplasmic reticulum and Golgi apparatus and eventually undergo post-translational modifications similar to those occurring in higher eukaryotes, proteins tend to be over-glycosylated when expressed in yeasts [10,11]
Comparison between constitutive and induced productions of Gap1 In order to assess the ability of our expression protocol to provide large quantities of the target protein, we selected a mutant version of Gap1, Gap19KR, where the first 9 lysines present in the cytosolic amino-terminal part are mutated into arginine in order to protect the protein against ubiquitylation, making it resistant to endocytosis and subsequent degradation without affecting its activity [22]

Summary

Introduction

Decades 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. Membrane proteins of known structure are mostly of prokaryotic origin, probably due to the difficulties associated with the expression of eukaryotic proteins in scalable systems. Membrane proteins destined to the plasma membrane traffic through the endoplasmic reticulum and Golgi apparatus and eventually undergo post-translational modifications similar to those occurring in higher eukaryotes, proteins tend to be over-glycosylated when expressed in yeasts [10,11]. A large number of expression vectors are available for protein production in S. cerevisiae and transformation-associated in vivo recombination in these vectors allows to test various plasmid constructs (harboring alternative gene promoters, tags, linkers, and eventually including mutations in the genes of interest). The biomass obtained from expressing cells can be lower than for other yeasts (such as Pichia pastoris), the relative amount of protein of interest versus the total biomass can result in better purification yield and purity [12]

Methods

Results

Conclusion