A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

Yeasts are attractive organisms for recombinant protein production. They combine highly developed genetic systems and ease of use with reductions in time and costs. We describe an autoselection system for recombinant protein expression in Saccharomyces cerevisiae which increases yields 5-10-fold compared to conditional selection for expression plasmids. Multicopy expression plasmids encoding essential MOB1 or CDC28 genes are absolutely necessary for the viability of host cells with mob1 or cdc28 deletions in their genomes. Such plasmids are stably maintained, even in rich medium, so optimising biomass production and yields of recombinant protein. Plasmid copy numbers are also increased by limiting selective MOB1 and CDC28 gene expression prior to induction. GST- or 6His-tagged proteins are produced for affinity purification and are expressed from a conditional GAL1-10 promoter to avoid potentially toxic effects of recombinant proteins on growth. Autoselection systems for expressing single or pairs of proteins are described. We demonstrate the versatility of this system by expressing proteins from a number of organisms and include several large and problematic products. The in vitro reconstruction of a step in mitotic regulation shows how this expression system can be successfully applied to the detailed analysis of complex metabolic pathways.     

Alpha-agglutinin expression in Saccharomyces cerevisiae

A polyclonal antiserum raised against purified alpha-agglutinin was made specific for alpha-agglutinin after adsorption with a cells. The adsorbed antiserum identified alpha-agglutinin peptides on Western blots and bound to cell surface alpha-agglutinin, inhibiting the binding of alpha cells to a cells. Using the antibody, we have determined that 1) the surface distribution of alpha-agglutinin on alpha cells is polar, 2) about 5 x 10(4) molecules/cell are constitutively expressed on strain X2180-1B (alpha) cells, and 3) treatment of alpha cells with the sex pheromone a-factor causes an increase in cell surface alpha-agglutinin, consistent with the a-factor induced increase in cell agglutinability.     

An innovative protein expression system using RNA polymerase I for large-scale screening of high-nucleic-acid content Saccharomyces cerevisiae strains

Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5′ caps, thus cannot be translated to proteins. To screen high-nucleic-acid content yeasts more efficiently, a cap-independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I-recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo-dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap-dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high-nucleic-acid content yeasts.     

Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multi-cloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.     

Dual system for potassium transport in Saccharomyces cerevisiae.

In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.     

A system for temperature-controlled expression of a foreign gene with dual mode in Saccharomyces cerevisiae

A temperature-controlled expression system for a foreign gene in Saccharomyces cerevisiae was constructed. In this system, a MATa hmlα2-102 HMRa sir3–8^ts double mutant was used as host, and a DNA fragment bearing the promoter and pre- or pre-pro regions of the MFα1 gene encoding the α-factor of S. cerevisiae was used as a promoter for expression of a foreign gene cloned on a vector. When the host cells were incubated at a restrictive temperature for the sir3–8^ts mutation (30°C to 35°C) they showed the α mating type and a PHO5 DNA fragment of S. cerevisiae, encoding repressible acid phosphatase, connected downstream of the MFα1 promoter was expressed. But when they were incubated at permissive lower temperature (25°C), at which they have the a mating type, the PHO5 DNA was not expressed. Acid phosphatase activity was increased 30-fold by shifting the incubation temperature from 25°C to 30°C. In this system it may also be possible to express a foreign gene at lower temperature but shut off its expression at higher temperature by connecting the gene to a promoter DNA of an a-specific gene.     

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.     

An edeine resistant mRNA-dependent protein synthesis system from a Saccharomyces cerevisiae mutant

A cell-free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2 microM edeine, while a similar system from wild-type strain was completely inhibited by the drug. The mutant ribosomes showed an affinity for [125I]edeine comparable to the wild-type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine-resistant capacity of the mutant.     

An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae

A new in vivo assay system has been developed to study programmed frameshifting in the yeast Saccharomyces cerevisiae. Frameshift signals are inserted between the Renilla and firefly luciferase reporter genes contained in a yeast expression vector and the two activities are directly measured from cell lysates in one tube. Similar to other bicistronic reporter systems, this one allows the efficient estimation of recoding efficiency by comparison of the normalized activity ratios from each luciferase protein. The assay system has been applied to HIV-1 and L-A directed programmed -1 frameshifting and Ty1 and Ty3 directed +1 frameshifting. The assay system is amenable to high-throughput screening.     

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His₆ tag fusion.The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the Berlin Protein Structure Factory. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).