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

Abstract

Background: 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.

Results: During 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.

Conclusions: Our 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.

Figure 1

Schematic representation of the vectors used during this work. They all derived from pRS416 and contain the same features: a yeast selection marker (URA3), an origin of replication for propagation in yeast (CEN/ARS), a marker of selection and an origin of replication for bacteria (ori and ampicillin resistance). All constructs were obtained by in vivo recombination.

Figure 2

Western blot showing the expression of Gap1-GST-6his developed with an antibody directed against penta-histidines. The gene is expressed under the regulation of its natural promoter (1, minimal medium) or under the regulation of the promoter of PMA1 (2, minimal medium; 3, rich medium). The same amount of total protein is loaded on each lanes. The stacking gel was also transferred and its limit is indicated.

Figure 3

Expression, purification and characterization of Gap1expressed using the nitrogen catabolite repression promoter system. A) Schematic representation of the medium switch protocol. B) Expression control of Gap1 by western blot revealed by an antibody against penta-histidines. Samples were taken just before the switch to the inductive medium (1), one hour after medium switch (2), 2 hours (3) and 23 hours just before harvesting the cells (4). C) Purity control by SDS-PAGE of Gap1 after purification using affinity chromatography. The protein is revealed by Coomassie staining. D) Infrared spectrum of reconstituted Gap1 in yeast lipid extract, peak of C = O bounds of lipids and proteins are highlighted.

Figure 4

Quantification and localization of MD-2, N-Gap1, Uga4, Gap1 and Vglut1 expressed using the nitrogen catabolite repression promoter system. A) Expression control by western blot done on total cellular extract after 24 and 48 hours of culture on inductive medium. The same amount of total protein (normalized by OD measurement) was loaded on each lanes. Proteins are revealed by an antibody against GFP. Expected sizes for the GFP-tagged proteins: MD2 (47.6 kDa), N-ter Gap1 (38.7 kDa), Uga4 (91.2 kDa), Gap1 (95.2 kDa), Vglut1 (90.9 kDa). B)In vivo fluorescence of expressing cells cultivated 24 hours on inductive medium. Untransformed cells have been used as negative control (−). All fluorescence pictures were taken using the same exposure time. C) Quantification of expression by GFP fluorescence measurements. Cells cultivated for 24 and 48 hours on inductive medium were submitted to GFP fluorescence measurements. Signal from non-induced cells was used for auto-fluorescence background subtraction. Fluorescence intensity were compared to a standard curve established using purified GFP. The quantification was calculated per gram of wet weight cells, corresponding to ~25 ml of fermenter culture at OD₆₆₀ 40.