Cerevisiae Expression System Research Articles

Characterization of GPAT gene family in Euphorbia lathyris L. and elucidating the role of ElGPAT9 in the biosynthesis of oils and pollen viability

Caper spurge (Euphorbia lathyris L.), known for its high content of oleic acid-enriched seed oils, has gained recognition as a valuable non-edible feedstock for biodiesel production. However, there is limited understanding of the biosynthesis and regulation of these desirable seed oils. In this study, an omics-based approach and genetic transformation were employed to investigate the glycerol-3-phosphate acyltransferase (GPAT) enzyme, which catalyzes the initial acylation reaction in triacylglycerol (TAG) biosynthesis, in caper spurge. Ten genes encoding ElGPAT, identified from the E. lathyris genome, were classified into three phylogenetic groups, exhibiting similar protein motifs and gene structures. RNA-seq data revealed distinct expression patterns of these ElGPAT genes in various tissues and different stages of seed development. Notably, ElGPAT9 showed the highest expression in flowers and developing seeds, with its encoded protein localized in the endoplasmic reticulum. Experiments using a yeast (Saccharomyces cerevisiae) expression system demonstrated that ElGPAT9 exhibited strong GPAT enzyme activity, crucial for TAG assembly, and preference for oleic acid (C18:1), as confirmed by feeding tests with exogenous fatty acids (FA). Similarly, stable transgenic lines of Nicotiana tabacum over-expressing ElGPAT9 showed increased levels of total oil and oleic acid, as well as improved pollen viability, without any growth penalty compared to wild-type and empty-vector controls. The transcriptome based WGCNA also identified genes related to lipid synthesis that are co-expressed with ElGPAT9. These findings provide a foundation for further understanding the roles of ElGPAT family members in E. lathyris and suggest the potential of ElGPAT9 as a desirable target for genetic engineering to enhance the production of vegetable oils enriched with C18:1 in caper spurge and other oil crops, contributing to sustainable biodiesel and lipid product production.

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

Expression of a Salt-Tolerant Pseudolysin in Yeast for Efficient Protein Hydrolysis under High-Salt Conditions.

Protease biocatalysis in a high-salt environment is very attractive for applications in the detergent industry, the production of diagnostic kits, and traditional food fermentation. However, high-salt conditions can reduce protease activity or even inactivate enzymes. Herein, in order to explore new protease sources, we expressed a salt-tolerant pseudolysin of Pseudomonas aeruginosa SWJSS3 isolated from deep-sea mud in Saccharomyces cerevisiae. After optimizing the concentration of ion cofactors in yeast peptone dextrose (YPD) medium, the proteolytic activity in the supernatant was 2.41 times more than that in the control group when supplemented with 5 mM CaCl2 and 0.4 mM ZnCl2. The extracellular proteolytic activity of pseudolysin reached 258.95 U/mL with optimized expression cassettes. In addition, the S. cerevisiae expression system increased the salt tolerance of pseudolysin to sodium chloride (NaCl)and sodium dodecyl sulfate (SDS) and the recombinant pseudolysin retained 15.19% activity when stored in 3 M NaCl for 7 days. The recombinant pseudolysin was able to efficiently degrade the β-conglycinin from low-denatured soy protein isolates and glycinin from high-denatured soy protein isolates under high temperatures (60 °C) and high-salt (3 M NaCl) conditions. Our study provides a salt-tolerant recombinant protease with promising applications in protein hydrolysis under high-salt conditions.

Expression of zinc transporter 8 in Saccharomyces cerevisiae and its antigenicity analysis

Zinc transporter 8 (ZnT8) is an important candidate antigen for type Ⅰ diabetes. The autoantibody detection kit based on ZnT8 can be used to help diagnose type Ⅰ diabetes, and the related products have been launched in Europe and the United States. Since the recombinant production system of active ZnT8 has not been established in China, this key raw material is heavily dependent on imports. We used Saccharomyces cerevisiae to carry out the recombinant expression of ZnT8. First, multiple antigenic forms of ZnT8 were designed as C-terminal haploid (C), C-terminal diploid (C-C), and N-terminal and C-terminal concatemers (N-C). The proteins were expressed, purified and tested for antigenicity by bridging-type ELISA. The serum of 13 patients with type Ⅰ diabetes and the serum of 16 healthy volunteers were detected. C, N-C, and C-C proteins had similar detection rates, which were 53.8% (7/13), 61.5% (8/13) and 53.8% (7/13). The specificity of the three groups was 100% (16/16). The detection value on positive samples P3, P4, and P8 increased by more than 90%, indicating better serum antibody recognition ability. Finally, N-C protein was selected for further serum sample testing, and the test results were characterized by receiver operating characteristic (ROC) curve for sensitivity and specificity. Compared with imported gold standard antigen, the sensitivity was 76.9% (10/13) and the specificity was 87.5% (14/16). There was no significant difference in the sensitivity of the method, but the specificity needed to be improved. In conclusion, the ZnT8 N-terminal and C-terminal concatemer protein developed based on S. cerevisiae expression system is expected to be a key alternative raw material in the development of in vitro diagnostic reagents for type Ⅰ diabetes.

Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae.

Structural and functional eukaryotic membrane protein research continues to grow at an increasing rate, placing greater significance on leveraging productive protein expression pipelines to feed downstream studies. Bacterial expression systems (e.g., E. coli) are often the preferred system due to their simple growth conditions, relative simplicity in experimental workflow, low overall cost per liter of cell growth, and ease of genetic manipulation. However, overproduction success of eukaryotic membrane proteins in bacterial systems is hindered by the limited native processing ability of bacterial systems for important protein folding interactions (e.g., disulfide bonds), post-translational modifications (e.g., glycosylation), and inherent disadvantages in protein trafficking and folding machinery compared to other expression systems.In contrast, Saccharomyces cerevisiae expression systems combine positive benefits of simpler bacterial systems with those of more complex eukaryotic systems (e.g., mammalian cells). Benefits include inexpensive growth, robust DNA repair and recombination machinery, amenability to high density growths in bioreactors, efficient transformation, and robust post-translational modification machinery. These characteristics make S. cerevisiae a viable first-alternative when bacterial overproduction is insufficient. Thus, this chapter provides a framework, using methods that have proven successful in prior efforts, for overproducing membrane anchored or membrane integrated proteins in S. cerevisiae. The framework is designed to improve yields for all levels of overexpression expertise, providing optimization insights for the variety of processes involved in heterologous protein expression.

Mapping human calreticulin regions important for structural stability

Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca2+ homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (Tm), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166–187 (“cluster 2”) for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit.Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).

In-vivo half-life and hypoglycemic bioactivity of a fusion protein of exenatide and elastin-based polypeptide from recombinant Saccharomyces cerevisiae

Exenatide (Ex) is a 39-amino acid peptide of glucagon-like peptide-1 (GLP-1) receptor agonist that was approved by the FDA in 2005 as a Type II diabetes treatment. It shows a 53% homology with GLP-1 but has an extended half-life (ca. 2.4 h) relative to GLP-1 (ca. 2–3 min). In this study, to further extend its in vivo half-life, we constructed a fusion protein (Ex-(EBP)10-6xHis) using a biocompatible and inert elastin-based polypeptide (EBP) as a fusion partner. Valine was inserted into the guest position of the pentapeptide (VPGXG), no linker sequence was inserted in between the EBPs, and (EBP)10-6xHis tag was attached to the C-terminus of exenatide. By using a recombinant Saccharomyces cerevisiae expression system, the fusion protein was expressed and secreted to the broth and purified by Ni-NTA affinity chromatography. Compared with the native exenatide, the physical half-life of the fusion protein was ca. 3.7-fold extended while approximately 72% of the in-vitro insulin secreting activity was maintained. However, the biological half-life measured by a glucose tolerance test (GTT) and the hypoglycemic test in mice was not significantly different from that of the native form. The effects of EBPylation on bioactivity and half-life of the fusion protein are similar to those of PEGylation. The result suggests that the bioactivity and half-life should be carefully balanced to obtain optimal fusion proteins. We expect that EBPylation using an optimal repeat number of EBP can be an alternative to chemical modification for therapeutic biobetters with extended half-life.

Secretory expression of β-mannanase in Saccharomyces cerevisiae and its high efficiency for hydrolysis of mannans to mannooligosaccharides.

Degradation of mannans is a key process in the production of foods and prebiotics. β-Mannanase is the key enzyme that hydrolyzes 1,4-β-D-mannosidic linkages in mannans. Heterogeneous expression of β-mannanase in Pichia pastoris systems is widely used; however, Saccharomyces cerevisiae expression systems are more reliable and safer. We optimized β-mannanase gene from Aspergillus sulphureus and expressed it in five S. cerevisiae strains. Haploid and diploid strains, and strains with constitutive promoter TEF1 or inducible promoter GAL1, were tested for enzyme expression in synthetic auxotrophic or complex medium. Highest efficiency expression was observed for haploid strain BY4741 integrated with β-mannanase gene under constitutive promoter TEF1, cultured in complex medium. In fed-batch culture in a fermentor, enzyme activity reached ~ 24U/mL after 36h, and production efficiency reached 16U/mL/day. Optimal enzyme pH was 2.0-7.0, and optimal temperature was 60°C. In studies of β-mannanase kinetic parameters for two substrates, locust bean gum galactomannan (LBG) gave Km = 24.13mg/mL and Vmax = 715U/mg, while konjac glucomannan (KGM) gave Km = 33mg/mL and Vmax = 625U/mg. One-hour hydrolysis efficiency values were 57% for 1% LBG, 74% for 1% KGM, 39% for 10% LBG, and 53% for 10% KGM. HPLC analysis revealed that the major hydrolysis products were the oligosaccharides mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose. Our findings show that this β-mannanase has high efficiency for hydrolysis of mannans to mannooligosaccharides, a type of prebiotic, suggesting strong potential application in food industries.

Cloning and expression of NS2B/NS3 protein of DENV3 Indonesia strain in Saccharomyces cerevisiae expression system for the development of Dengue vaccine

NS3 protein is 618 amino acids (aa) in length containing serine protease and helicase domains required for DENV replication. Alignment of consensus amino acid sequences from all four DENV serotypes demonstrated that this protein is more conserved (78%) among the different dengue serotypes, which elicits a strong cellular immune response after viral infection in humans and animal models. Present study, a central hydrophilic region of NS3 cofactor, NS2B (NS2BH) with full length of NS3 genes DENV3 Indonesian strain were amplified from CDNA following PCR, and inserted to PYES2/CT shuttle vector. The recombinant plasmid was transformed and expressed in Saccharomyces cerevisiae expression system. As result, detection with Anti-His detector and Anti-NS3 shown NS2BH/NS3 was expressed as 83 KDA protein band. We found that addition of NS2BH on NS3 full length construction plasmid increase the yield and activity of protein expression in S. cerevisiae. In future study, our recombinant NS2B/NS3 protein can be used as recombinant protein in Dengue vaccine development.

Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein.

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein's molecular mechanism through the combination of structure-function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure-function studies on dynein.

Construction and verification of CYP3A5 gene polymorphisms using a Saccharomycescerevisiae expression system to predict drug metabolism.

The present study evaluated the ability of a Saccharomyces cerevisiae expression system to predict the pharmacokinetic (PK) activity of a calcium channel blocker in patients with distinct cytochrome P450 3A5 (CYP3A5) polymorphisms. The blood pressure lowering activity of amlodipine in 57 hypertensive patients with CYP3A5*1/*1, CYP3A5*1/*3, CYP3A5*4 and CYP3A5*6 polymorphisms was evaluated by the current study. Subsequently, a Saccharomyces cerevisiae expression system for CYP3A5 gene polymorphisms was constructed to examine the PK activity of CYP3A5*1/*1, CYP3A5*4 and CYP3A5*6 polymorphisms. This system was used to predict the PK of amlodipine and was compared with the in vivo data from different gene polymorphism groups. The blood pressure lowering activity of amlodipine in hypertensive patients varied among CYP3A5 polymorphisms. The in vivo results demonstrated that CYP3A5*6 exhibited the highest metabolic rate, followed by CYP3A5*1/*1, CYP3A5*4 and CYP3A5*1/*3. The difference between CYP3A5*6 and CYP3A5*1/*1 was not statistically significant (P=0.5). In accordance with in vivo data, CYP3A5*1/*1 exhibited the highest in vitro metabolic rate, followed by CYP3A5*6 and CYP3A5*4. With the exception of the comparison between CYP3A5*6 and CYP3A5*1/*1, polymorphisms exhibited statistically significant differences compared with CYP3A5*1/*1 (P<0.05). The Saccharomyces cerevisiae expression system may be a cost effective and potentially useful tool for assessing the PK activity of drugs that are metabolized by CYP3A5.

Yeast as a Heterologous System to Functionally Characterize a Multiple Rust Resistance Gene that Encodes a Hexose Transporter.

Recently, the Lr67 resistance gene was identified as a hexose transporter variant which confers adult plant rust and mildew resistance in wheat. Methodologies used to characterize the protein encoded by Lr67 may be of use to non-transporter experts conducting similar experiments with other hexose transporters. Hence, in this chapter, we detail a protocol for the functional characterization of hexose transporter proteins in the Saccharomyces cerevisiae expression system. We also provide guidance on the use of metabolic inhibitors and competing sugars to probe transporter structural features, energization, and specificity.

Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome.

BackgroundTrypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome.MethodsThe functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy.ResultsIn the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (Km = 0.085 ± 0.04 mM) and low affinity for ornithine (Km = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [3H]-arginine displayed a 16-fold higher efflux of [3H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles.ConclusionsTcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.

The Optimization of Saccharomyces cerevisiae Expression System by Mutagenesis of Promoter and Host Strain

An expression system with high efficiency is very important for recombinant proteins production in biopharmaceutical field. Saccharomyces cerevisiae is a food-graded eukayotic organism. The features of short generation time,simple culture condition and well-characterized manipulation techniques make yeast S. cerevisiae an attractive cell factory for production of heterologous protein. Here,for the purpose to improve the efficiency of p HR expression system which was constructed in our lab previously.,the promoter of p HR expression vector( PTEF) and host strain Y16 were modified by the way of error-prone PCR and mutagenesis respectively. After several rounds of screening,a mutant PTEFv1 with higher efficiency than the mother promoter PTEFwas obtained.Two modified yeast strains Y16-E14 and Y16-E19 were identified with higher productivity of heterologous protein than yeast Y16. Then PTEFv1 and yeast Y16-E14 were used to construct the novel p HR-N expression system. To evaluate the ability of p HR-N expression system,yeast green fluorescent protein( GFP) and human serum albumin( HSA) were chosed to be expressed intracellularly and extracellularly respectively. The results showed that p HR-N system had higher ability to produce either intracellular GFP or extracellular HSA than p HR system.The p HR-N yeast expression system provides a valuable resource for future application in recombinant protein production.

Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

BackgroundPorcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.ResultsIn this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.ConclusionsWe have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.

Biochemical characterization of a functional recombinant aryl-alcohol dehydrogenase from Taiwanofungus camphorata.

BackgroundAryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds.ResultsOne TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied.ConclusionsThe coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-14) contains supplementary material, which is available to authorized users.

Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.

Albusin B, mass‐produced by the Saccharomyces cerevisiae suppression system, enhances lipid utilisation and antioxidant capacity in mice

Albusin B (bacteriocin), isolated from Ruminococcus albus 7 and mass-produced by the Saccharomyces cerevisiae expression system, has previously been shown to have a beneficial effect on lipid metabolism in broiler chickens. The present study was focused on the effect of albusin B on lipid metabolism in mice and the potential of albusin B-expressing yeast product (albusin B) as a food supplement. Forty-five BALB/c male mice at 6 weeks of age were each orally administered normal saline (control), yeast (0.125 mg kg(-1) ) or albusin B (0.125 mg kg(-1) ) for 14 days and then euthanised. Compared with the control group, albusin B-fed mice exhibited decreased body weight and plasma levels of triglycerides and free fatty acids but increased plasma high-density lipoprotein. Albusin B-fed mice showed higher mRNA expression of fatty acid oxidation in the ileum, heart and liver than control mice. Compared with the control treatment, both yeast and albusin B treatments caused a decrease in mRNA expression of fatty acid synthesis in the heart and liver. Moreover, albusin B suppressed mRNA levels of lipogenesis in the ileum and liver. Albusin B-fed mice exhibited more favourable adenosine triphosphate production and antioxidant capacity in the heart and liver. Albusin B treatment led to a significantly lower respiratory quotient than that of the control, whereas yeast treatment did not. This study demonstrated a beneficial effect of albusin B on lipid utilisation and anti-atherosclerotic and antioxidant capacities in mice. However, more comprehensive studies are required to elucidate the exact mechanism behind the effect of albusin B.

Systematic analysis of a dipeptide library for inhibitor development using human dipeptidyl peptidase IV produced by a Saccharomyces cerevisiae expression system

The inhibition of human dipeptidyl peptidase IV/CD26 (hDPPIV) is an accepted treatment for type 2 diabetes. In this study, an extracellular production system of hDPPIV using Saccharomyces cerevisiae was established to facilitate the screening of hDPPIV inhibitors. As dipeptides that mimic the hDPPIV substrate are candidate inhibitors of this protein, X–Ala or X–Pro dipeptides (in which X represents any amino acid) were tested systematically. Based on the results obtained in the first screening, a second screening was performed for Trp–X dipeptides. To elucidate the manner via which the physicochemical features at the P1 and P2 positions contributed to the hDPPIV inhibitory effect, correlations between the inhibitory activity of dipeptides and 13 amino acid indices were analyzed. The most effective inhibitory dipeptide was Trp–Pro (Ki=0.04mM). The mode of inhibition of hDPPIV by dipeptides was explained well by some amino acid indices and by the structure of the substrate-binding site of hDPPIV. The information obtained from the systematic analysis of a dipeptide library provides important clues for the development of hDPPIV targeting drugs and functional foods for type 2 diabetes.

Production and purification of theVP1 capsid protein of a novel canine norovirus using the Saccharomyces cerevisiae expression system

The discovery of a novel canine norovirus was recently reported. The VP1 capsid protein of this canine norovirus was expressed in Saccharomyces cerevisiae and purified with a yield of 1.6–1.8mg/L. The yeast-expressed VP1 protein self-assembled into virus-like particles and exhibited antigenicity, proving to be suitable for serological assays.