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Results for “Mating Factor”

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At least 343 records · Page 19Linked to original sources

PubMed1988

A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1.

Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.

Amino Acid Sequence↗

PubMed2000

Sequence and expression of Thai Rosewood beta-glucosidase/beta-fucosidase, a family 1 glycosyl hydrolase glycoprotein.

Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic beta-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-alpha-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.

Amino Acid Sequence↗

PubMed1991

Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae.

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.

Amino Acid Sequence↗

PubMed1996

A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain.

A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain. The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae. The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l. The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences. Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant. Two different types of affinity chromatography were used to purify either propapain or papain. The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously. The kinetic parameters obtained agree well with the literature values. The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems. This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.

Amino Acid Sequence↗

PubMed2006

Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion.

In fish, FSH is generally important for early gonadal development and vitellogenesis. As in mammals, FSH is a heterodimer composed of an alpha subunit that is noncovalently associated with the hormone-specific beta subunit. The objective of the present study was to express glycosylated, properly folded, and biologically active tilapia FSH (tFSH) using the Pichia pastoris expression system. Using this material, we aimed to develop a specific ELISA and to enable the study of FSH response to GnRH. The methylotrophic yeast P. pastoris was used to coexpress recombinant genes formed by fusion of mating factor alpha leader and tilapia fshb and cga coding sequences. Western blot analysis of tilapia pituitary FSH, resolved by SDS-PAGE, yielded a band of 15 kDa, while recombinant tFSH beta (rtFSH beta) and rtFSH beta alpha had molecular masses of 17-18 kDa and 26-30 kDa, respectively. Recombinant tFSH beta alpha was found to bear only N-linked carbohydrates. Recombinant tFSH beta alpha significantly enhanced 11-ketotestosterone (11-KT) and estradiol secretion from tilapia testes and ovaries, respectively, in a dose-dependent manner (similar to tilapia pituitary extract, affinity-purified pituitary FSH, and porcine FSH). Using antibodies raised against rtFSH beta, FSH-containing cells were localized adjacent to hypothalamic nerve fibers ramifying in the proximal pars distalis (PPD), while LH cells were localized in a more peripheral region of the PPD. Moreover, FSH is under the control of hypothalamic decapeptide GnRH, an effect that was abolished through the use of specific bioneutralizing antisera, anti-rtFSH beta. It also reduced basal secretion of 11-KT.

Animals↗

PubMed2004

Expression of functionally active helper component protein of Tobacco etch potyvirus in the yeast Pichia pastoris.

Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro). To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used. A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae alpha-mating factor secretory peptide coding region, was inserted into the P. pastoris genome using a modified version of the pPIC9 vector. The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector. Our results indicate that P. pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assays.

Amino Acid Sequence↗

PubMed2005

High level expression of a recombinant acid phytase gene in Pichia pastoris.

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.

6-Phytase↗

PubMed1991

Production and purification of recombinant human interleukin-6 secreted by the yeast Saccharomyces cerevisiae.

The coding region of the human interleukin-6 (hIL6) gene was fused to the prepro secretion signal of the alpha-mating factor gene in several yeast host strains. It was found that the KEX-2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL6 sequence, but that unspecific cleavage of the precursor protein had occurred. The prepro-Lys-Arg-Ala-Pro-IL6 sequence, however, was correctly recognized and cleaved by the KEX-2 protease, and IL6 was efficiently secreted into the culture medium. The N-terminal Ala-Pro peptide was removed during processing by wild-type yeast strains, but was retained in a ste13 mutant. IL6 as well as the aberrant proteins were not glycosylated. The transformed cells could secrete up to 30 micrograms/ml IL6. The protein was purified from the medium to homogeneity by ion-exchange chromatography and gel filtration, and had a specific activity of about 2 x 10(8) IU/mg in a proliferation assay.

Amino Acid Sequence↗

PubMed1997

Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase.

The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues. N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment. Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.

Animals↗

PubMed1991

Expression in yeast of a cDNA clone encoding a transmembrane glycoprotein gp41 fragment (a.a. 591-642) bearing the major immunodominant domain of human immunodeficiency virus.

A cDNA clone corresponding to the gp41 gene fragment nucl. 7573-7730 of the human immunodeficiency virus type 1 (HIV-1) was selected from a random HIV-1 genomic library expressed in yeast. This clone encodes a 52-residue long peptide (amino acid (a.a.)) 591-642) bearing the major immunodominant domain (a.a. 598-609) of the HIV-1 transmembrane glycoprotein gp41. Expression of the recombinant peptide pSE-env591-642 was driven by the alpha-mating factor leader sequence contained in a plasmid pSE-x allowing the synthesis and secretion of foreign gene product in Saccharomyces cerevisiae. Time-course analysis of the secretion into culture medium revealed an optimal production of the glycoprotein fragment at 28-30 h with no observable cytotoxicity. The secreted peptide is highly glycosylated with NH2-terminal heterogeneity probably due to different post-translational modifications. The secreted peptide shows an extreme antigenicity since in ELISA assays, as few as 5 microliters/well of crude supernatant are sufficient to obtain a strong detection by monoclonal antibodies or by 100% of sera from HIV-infected individuals. The purified glycopeptide pSE-env591-642 binds to a monoclonal antibody directed against the immunodominant epitope (a.a. 603-609) with an affinity similar to that of the complete glycoprotein gp160 (Kd values within the 10(-10) M range) and with a 100-fold higher affinity than that of a linear peptide fragment SP-env584-609. These results indicate that overexpression in yeast can efficiently provide an abundant source of highly antigenic gp41 protein fragment pSE-env591-642 which retains the antigenic properties of the native gp160 protein. Such a recombinant peptide should therefore be considered as a good candidate for antigen in HIV detection tests.

Amino Acid Sequence↗

PubMed1990

An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control.

A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2), is thought to be an early intermediate in an insulin-stimulated phosphorylation cascade and in a variety of other mammalian cell responses to extracellular signals. A complementary DNA that encodes this protein serine-threonine kinase has been cloned, and the protein designated extracellular signal-regulated kinase 1 (ERK1). ERK1 has striking similarity to two protein kinases, KSS1 and FUS3, from yeast. The yeast kinases function in an antagonistic manner to regulate the cell cycle in response to mating factors. Thus, ERK1 and the two yeast kinases constitute a family of evolutionarily conserved enzymes involved in regulating the response of eukaryotic cells to extracellular signals.

Amino Acid Sequence↗

PubMed2001

Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes.

The beta-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor alpha1 signal peptide (MFalpha1(s)) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant beta-xylosidase showed optimum activity at 60 degrees C and pH 3.2 and optimum stability at 50 degrees C. The K(i(app)) value for D-xylose and xylobiose for the recombinant beta-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 beta-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of beta-xylanase activity when expressing only the beta-xylanase and 860 nkat/ml when coexpressing the beta-xylanase with the beta-xylosidase. The maximum beta-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the beta-xylanase. Coproduction of the beta-xylanase and beta-xylosidase enabled S. cerevisiae to degrade birchwood xylan to D-xylose.

Aspergillus niger↗

PubMed1975

Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae.

The interdependence of spindle plaque with other aspects of cell division and conjugation in Saccharomyces cerevisiae has been investigated. Three forms of the spindle plaque appear sequentially before the formation of the complete, intranuclear spindle. The single plaque is present initially in the mitotic cycle; it becomes transformed into a satellite-bearing single plaque during the latter part of G1. Subsequently, plaque duplication yields the double plaque characteristic of the early phase of budding, which coincides with the period of chromosome replication (S). The eventual separation of these plaques to form a complete spindle, with a single plaque at each pole, is nearly coincident with the completion of S. The form of the plaque differs in two independent cases of G1 arrest: the single plaque is found in a cell in stationary arrest of growth, whereas a cell arrested by mating factors in preparation for conjugation contains a satellite-bearing single plaque. The latter form is retained during zygote formation, where it serves as the initial site of fusion of each prezygotic nuceus with the other. This fusion results in the formation of a single zygotic nucleus with a satellite-bearing single plaque, which is subsequently transformed into a double plaque as the zygote buds. The double plaque is situated adjacent to the site of bud emergence in both vegetative cells and zygotes.

Cell Division↗

PubMed1977

Peptide transport in yeast: utilization of leucine- and lysine-containing peptides by Saccharomyces cerevisiae.

A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomyces cerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leucine were not utilized by the mutant. Cellular peptidases released leucine or lysine from all of these non-growth-supporting peptides, suggesting that the failure of strain Z1-2D to utilize these compounds reflects their failure to enter the yeast. Competition studies employing phenylalanine or non-leucine-containing peptides showed that the uptake of peptides into S. cerevisiae Z1-2D is distinct from that of amino acids and that di- and oligopeptides may share a common transport system. The failure of strain Z1-2D to utilize any peptide larger than (Leu)3 may indicate a transport size limit. Such a size limit would influence the construction of models that explain the action of yeast mating factors.

Biological Transport, Active↗

PubMed1994

G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover.

The 545-residue Cln2 protein, like the other G1 cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G1 cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK delta 40). The resulting chimeric protein (hTK delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G1 arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28ts and cdc34ts mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2 PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.

Amino Acid Sequence↗

PubMed1986

Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication.

An electron microscopic study was made of the replication of rDNA chromatin of Saccharomyces cerevisiae. Two different methods were used to synchronize cells. cdc7-1 cells were raised to a restrictive temperature, whereas A364a cells were blocked with mating factor. Replication bubbles typically opened in the nontranscribed spacers of rDNA repeats in both cell types. The mean position of the center of these bubbles corresponds closely to a position where an autonomously replicating sequence previously has been mapped in an rDNA repeat. Clusters of replication bubbles containing up to four bubbles spaced one to three genes apart were seen opening in early S phase.

Chromatin↗

PubMed1989

Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product.

Mutations in the Schizosaccharomyces pombe ral2 gene cause a phenotype indistinguishable from that of the ras1-defective mutant. Using cloned ral2 DNA, we disrupted the chromosomal gene. The disruptants showed the same phenotype as the original ral2 isolates, i.e., they had spherical cells, had no detectable mating activity, and exhibited no response to the mating pheromone, but their vegetative growth was apparently normal. Sequence analysis of the ral2 gene suggests that it encodes a polypeptide of 611 amino acid residues whose predicted amino acid sequence shows no strong homology to any known protein. Either multiple copies or even a single copy of the ras1Val-17 allele, which is an activated form of ras1, restored rodlike cell morphology and ability to respond to the mating factor to ral2 mutants. These results suggest that the ral2 and ras1 gene products interact intimately and that the ral2 gene product is involved in activation of the ras1 protein in S. pombe.

Alleles↗

PubMed1989

The ras oncogene--an important regulatory element in lower eucaryotic organisms.

The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein. This gene is frequently activated in human cancer. As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied. In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism. Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle. Although analysis of RAS biochemistry in S. cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism. In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells. However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism. The identification of other GTP-binding proteins in S. cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.

Adenylyl Cyclases↗