A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

Peptides from the SARS-associated coronavirus as tags for protein expression and purification

Protein tagging with a peptide is a commonly used technique to facilitate protein detection and to carry out protein purification. Flexibility with respect to the peptide tag is essential since no single tag suites all purposes. This report describes the usage of two short peptides from the SARS-associated coronavirus nucleocapsid (SARS-N) protein as protein tags. Plasmids for the generation of tagged proteins were generated by ligating synthetic oligonucleotides for the peptide-coding regions downstream of the protein coding sequence. The data show recognition of prokaryotically expressed HIV-1 Gag/p24 fusion protein by Western blot and efficient affinity purification using monoclonal antibodies against the tags. The SARS peptide antibody system described presents an alternative tagging opportunity in the growing field of protein science.     

The Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein: one-step purification and functional analysis in vitro

Genetic expression versus plasmidic overexpression of a functional recombinant fusion protein combining the yeast Saccharomyces cerevisiae mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p) has been investigated, with the main aim of increasing the polar surface of the carrier to improve its crystallization properties. The gene encoding the his6-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2 or under the control of the strong yeast PMA1 promoter. In both cases, the chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, demonstrating its transport activity. Nevertheless, when the expression vector was used, the level of expression of Anc2-Cyc1(His6)p was no greater than that of the chimeric carrier obtained in yeast mitochondria after homologous recombination. Optimal conditions to extract and to purify Anc2-Cyc1(His6)p were determined. A series of detergents was screened for their ability to extract and to preserve in vitro the chimeric carrier. A rapid, single step purification of Anc2-Cyc1(His6)p was developed, using n-dodecyl-beta-d-maltoside (DoDM) as the best detergent to solubilize the chimeric protein. Carboxyatractyloside- (CATR-) and nucleotide-binding sites were preserved in the purified protein. Moreover, the Cyc1p moiety of Anc2-Cyc1(His6)p-CATR complex solubilized in DoDM was still able to interact in vitro with the cytochrome c oxidase (COX), with the same affinity as yeast Cyc1p. Improved production and purification of Anc2-Cyc1(His6)p-CATR complex opens up new possibilities for the use of this protein in crystallographic approaches to the yeast ADP/ATP carrier. Furthermore, Anc2-Cyc1(His6)p may be an useful molecular tool to investigate in vivo interactions between components of the respiratory chain complexes such as COX and the proteins implicated in ATP biogenesis, such as the ATP/ADP carrier.     

Expression and purification of homogenous proteins in Saccharomyces cerevisiae based on ubiquitin-FLAG fusion

The construction of an expression vector for increased expression of cytoplasmic proteins in Saccharomyces cerevisiae is described. To enhance the yield of expressed proteins, fusion of ubiquitin to an octapeptide (a FLAG tag) upstream of the respective model genes was applied. During protein maturation ubiquitin is efficiently removed by yeast autologous hydrolases, generating the FLAG octapeptide at the N-terminus. Fusion proteins were recognized by the specific monoclonal antibody M1 directed against the FLAG tag. The FLAG-tagged proteins were purified to homogeneity by immunoaffinity chromatography using an anti-FLAG M1 agarose. Different model proteins, green fluorescent protein, green fluorescent protein-human lysozyme, green fluorescent protein elongation-initiahon factor 5a, green fluorescent protein-rapamycin-selective 25-kDa immunophilin, and green fluorescent protein-heat shock protein 90 beta have been selected to demonstrate the efficiency of the new vector construct.     

Thermus thermophilus-derived protein tags that aid in preparation of insoluble viral proteins

The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 (T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.     

Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates

Affinity capture methods are widely used for isolation and analysis of protein complexes. Short peptide tags fused to the protein of interest normally facilitate straightforward purification and detection of interacting proteins. We investigated the suitability of applying C-terminally hexahistidine-tagged interleukin-12 (IL-12) alpha- and beta-chains as "bait" proteins for cocapturing novel binding partners using heterologous recombinant human embryonic kidney-293 (HEK-293) cell lines. The beta-chain, but not the alpha-chain, extracted from cell lysates was capable of binding to the Ni(2+)-nitrilotriacetic acid affinity resin under nondenaturing conditions. Retention of the alpha-chain on this matrix was dependent on treatment of cell lysates with high concentrations of chaotropes such as urea. Since under these conditions any noncovalent protein associations are destroyed, prior cross-linking of proteins interacting with the alpha-chain in intact cells was required. The use of the thiol-cleavable cross-linker 3,3'-dithiobis(succinimidyl proprionate) facilitated dissociation of alpha-chain-binding proteins by means of dithiothreitol following purification. Using this approach we were able to demonstrate a strong interaction between the endoplasmic reticulum chaperone calreticulin (CRT) and the IL-12 alpha-chain that was confirmed in a reciprocal anti-CRT immunoprecipitation assay. The assay presented here provides a simple approach to exposing concealed hexahistidine tags while retaining native noncovalent protein interactions and should be generally applicable in a range of pull-down or affinity capture methods aiming at analysis of protein complexes.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Expression of functional multidrug-resistance protein 1 in Saccharomyces cerevisiae: effects of N- and C-terminal affinity tags

Studies of the multidrug-resistance protein 1 (MRP1) have been hampered by the lack of a simple expression system allowing for rapid generation of mutants and yielding milligram amounts of protein. Here, we describe a Saccharomyces cerevisiae expression system that meets those conditions. MRP1 was expressed under the control of the constitutive PMA1 (yeast proton pump) promoter. The best conditions for expression were determined, including the use of the chemical chaperone glycerol, which increased MRP1 expression. N-terminal poly-histidine or FLAG affinity tags reduce MRP1 expression, whereas the same tags fused to the C-terminus had no effect. All the fusion proteins were functional. We conclude that because of its low cost and simplicity, the S. cerevisiae-based MRP1-expression system will be useful for studies where a large number of mutants or milligram amounts of purified MRP1 are needed.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.     

Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein

We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites.     

The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.     

Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Use of selected bacteria and yeast to protect gnotobiotic Artemia against different pathogens

To evaluate the potential probiotic effect of two bacterial strains towards Artemia cultured in different gnotobiotic conditions, challenge tests were performed with a virulent Vibrio campbellii or with an opportunistic Vibrio proteolyticus strain. For that purpose, three feed sources (different isogenic Saccharomyces cerevisiae mutant strains) were chosen, yielding distinct Artemia culture performances. Both bacterial strains, selected from previous well-performing Artemia cultures, were able to protect against the opportunistic V. proteolyticus, while, generally, these bacteria could not protect Artemia against V. campbellii. The quality of the feed provided (in the form of the isogenic mnn9 yeast mutant) to Artemia had a stronger influence on nauplii protection against the opportunistic and the virulent Vibrio than the addition of beneficial bacteria. This feed has a higher nutritional value for Artemia, but contains also more cell wall bound β-glucans and chitin. Data suggest that the change in the cell wall composition, rather than the overall better nutritional value, of the mnn9 strain is responsible for the protection against both Vibrios.     

A simple and effective set of PCR-based molecular markers for the monitoring of the Saccharomyces cerevisiae cell population during bioethanol fermentation

One of the defining features of the fermentation process used in the production of bioethanol from sugarcane feedstock is the dynamic nature of the yeast population. Minisatellite molecular markers are particularly useful for monitoring yeast communities because they produce polymorphic PCR products that typically display wide size variations. We compared the coding sequences derived from the genome of the sugarcane bioethanol strain JAY270/PE-2 to those of the reference Saccharomyces cerevisiae laboratory strain S288c, and searched for genes containing insertion or deletion polymorphisms larger than 24 bp. We then designed oligonucleotide primers flanking nine of these sites, and used them to amplify differentially sized PCR products. We analyzed the banding patterns in the most widely adopted sugarcane bioethanol strains and in several indigenous yeast contaminants, and found that our marker set had very good discriminatory power. Subsequently, these markers were used to successfully monitor the yeast cell populations in six sugarcane bioethanol distilleries. Additionally, we showed that most of the markers described here are also polymorphic among strains unrelated to bioethanol production, suggesting that they may be applied universally in S. cerevisiae. Because the relatively large polymorphisms are detectable in conventional agarose gels, our method is well suited to modestly equipped on-site laboratories at bioethanol distilleries, therefore providing both cost and time savings.     

Production of recombinant proteins by yeast cells

Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed.     

Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.     

Enhanced expression,detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags

The creation of a double His-tag fusion that forms a RNA stem loop in the mRNA encoding the N-terminus of the target protein is a novel approach for the enhancement of expression, purification, and detection of a recombinant protein. Compared to a single His-tag fusion, a tandem His-tag fusion RNA stem loop, located downstream of the constitutive groE and Ch promoters, enhanced heterologous gene expression in Brucella, Salmonella, and Escherichia. We demonstrated one-step detection and purification of recombinant green fluorescence protein (GFP) directly from Brucella spp. without using Escherichia coli as an expression host. The amount of purified GFP using the tandem His-tag RNA stem loop increased more than threefold; moreover, the sensitivity of detection increased more than fourfold in comparison to the single His-tag fusion form. This method has the potential to significantly improve heterologous gene expression and high-throughput protein synthesis and purification.     

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium

The human tumour suppressor P53 is a key protein involved in tumour suppression. P53 acts as a "guardian of genome" by regulating many target genes involved in cell cycle regulation, DNA repair and apoptosis. We report the P53 expression by the methylotrophic yeast Pichia pastoris using the methanol inducible AOX1 promoter. We have produced the rP53 in intracellular form as well as secreted using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence in two genetic contexts of Pichia, Mut(s) and Mut(+). The intracellular P53 was successfully produced by Mut(s) (KM71) as well as Mut(+) (X33) strains, however, the secreted form was mainly observed in the Mut(s) strain, despite a higher number of p53 copies integrated in the Mut(+) strain. Interestingly, in Mut(s) phenotype, the medium pH influences markedly the rP53 production since it was higher at pH 7 than 6.     

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.