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At least 271 records · Page 15Linked to original sources
PubMed1994

Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae.

We have tried to optimize the galactose-inducible gene expression system for the overproduction of the potent thrombin-specific inhibitor, hirudin, in a genetically engineered yeast, Saccharomyces cerevisiae. The expression and secretion of hirudin were directed by the galactose-inducible promoter, GAL10, and the mating factor alpha pre-pro leader sequence. The initial hirudin expression level in shake-flask culture was 2.3 mg 1-1. Modification of the expression vector and optimization of culture conditions, including the induction conditions, improved the level of hirudin gene expression and secretion into the culture supernatant more than 20-fold (50 mg 1-1) in a 4-1 scale batch cultivation. The expression and secretion level of hirudin seemed to be partially dependent on cell growth when galactose was used as a carbon source. Overexpression of the transcriptional activator, GAL4, appeared to have only negative effects on the expression of the hirudin gene and lacZ directed by the GAL10 promoter in the strain used in this study, unlike the previously reported examples. The complex medium containing yeast extract used for the increase of the cell mass and hirudin level did not show any detrimental effect on plasmid stability and did not complicate the downstream purification of hirudin from the culture supernatant. Moreover, the complex medium could greatly improve the hirudin productivity and reduce the degradation of hirudin produced in the culture supernatants.

Animals↗
PubMed1976

The conditions required for the induction of petite yeast mutants by fluorinated pyrimidines.

Cytoplasmic petite mutagenesis by 5-fluorouracil (5FU) was prevented by temperature sensitive mutations which blcoked either nuclear transcription or cytoplasmic translation. However, 5FU was also ineffective in resting cells and in cells exposed to alpha-mating factor, showing that cell division or nuclear DNA synthesis is required for the mutagenic event to take place. In addition, the mutagenic effect of 5FU was completely prevented by daunomycin, and since this agent preferentially inhibits respiratory growth and was shown to selectively block RNA synthesis in the mitochondria, it was concluded that petite mutagensis resulted from incorporation of 5FU into mitochondrial RNA. Since inhibition of mitochondrial protein synthesis by erythromycin had little immediate effect on the mutagenicity of 5FU, it was deduced that the RNA in question is not directly involved in mitochondrial translation, and may have a regulatory function.

Daunorubicin↗
PubMed1991

High-level secretion of biologically active aprotinin from the yeast Pichia pastoris.

A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.

Amino Acid Sequence↗
PubMed2006

High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris.

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6'-phytase, which owns high specific activity. To achieve a high expression level of 6'-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml(-1). The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50 degrees C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.

6-Phytase↗
PubMed2000

Coexpression of the Bacillus pumilus beta-xylosidase (xynB) gene with the Trichoderma reesei beta xylanase 2 (xyn2) gene in the yeast Saccharomyces cerevisiae.

The xynB gene encoding the Bacillus pumilus beta-xylosidase was expressed separately and jointly with the Trichoderma reesei beta-xylanase (xyn2) gene in the yeast Saccharomyces cerevisiae. Both genes were placed under the transcriptional control of the glucose-derepressible alcohol dehydrogenase 2 promoter (ADH2p) and terminator (ADH2T) sequences. The xynB gene was fused in frame to the yeast mating factor alpha1 secretion sequence (MFalpha1s) to effect secretion in S. cerevisiae. The fusion protein was designated Xlo1. Xlo1 produced in S. cerevisiae exhibited low affinity for xylobiose, but eventually hydrolyzed xylobiose and xylotriose to the monomeric constituent, D-xylose. Coproduction of Xyn2 and Xlo1 by S. cerevisiae led to a 25% increase in the amount of reducing sugars released from birchwood xylan compared to S. cerevisiae producing only the Xyn2 beta-xylanase. However, no D-xylose was produced from birchwood xylan, presumably due to very low Xlo1 beta-xylosidase activity and its low affinity for xylobiose.

Alcohol Dehydrogenase↗
PubMed2004

Effects of mutations in the N terminal region of the yeast G protein alpha-subunit Gpa1p on signaling by pheromone receptors.

The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the Galpha subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of Galpha as well. We screened for mutations in the N-terminal region of the Galpha subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast alpha-mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones alpha-factor and a-factor without affecting cellular levels of the subunit or its ability to interact with beta and gamma subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast Galpha subunit does not undergo extensive interactions with the alpha-factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.

Alleles↗
PubMed2006

Pichia pastoris is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon Pyrococcus abyssi.

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence alpha-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3'-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.

Archaeal Proteins↗
PubMed2005

cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds.

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal alpha-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.

Amino Acid Sequence↗
PubMed1996

Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2' positions.

The yeast aspartic protease Yap3 is localised to the secretory pathway and correctly cleaves pro-alpha-mating factor at its dibasic sites. We determined the specificity of Yap3 for mono-, di-, and multi-basic cleavage sites in the context of 15 residue synthetic proalbumin peptides. Yap3 cleaved after dibasic ArgArg and LysArg sites but not after monobasic Arg sites even when there was an additional arginine at -6 and/or -4. Yap3 did not cleave a tetra-arginine site and tri-basic sites (RRR and RRK) were poor substrates. Cleavage always occurred C-terminal to the last arginine in the di- or tri-basic sequence. The optimal cleavage site sequence was RR DR and this substrate was cleaved 8-9-fold faster than the normal RR DA sequence. In contrast to Kex2, Yap3 did not remove the propeptide from normal proalbumin or a range of natural or recombinant proalbumin variants. However at pH 4.0 Yap3 slowly cleaved proalbumin and albumin between domains 2 and 3.

Amino Acid Sequence↗
PubMed1996

Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris.

The human mu-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae alpha-mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase-1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell). Competition studies with non-selective, and mu-, delta- and kappa-selective opioid agonists and antagonists revealed a typical mu-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Cell Membrane↗
PubMed1989

Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae.

We have examined the roles of eukaryotic DNA topoisomerases I and II in DNA replication by the use of a set of four isogenic strains of Saccharomyces cerevisiae that are TOP1+ TOP2+, TOP1+ top2 ts, delta top1 TOP2+, and delta top1 top2 ts. Cells synchronized by treatment with the alpha-mating factor, or by cycles of feeding and starvation, were released from cell-cycle arrest, and the size distribution of DNA chains that were synthesized after the cells reentered the S-phase was determined as a function of time. The results indicate that synthesis of short DNA chains several thousand nucleotides in length can initiate in the absence of both topoisomerases, but their further elongation requires at least one of the two topoisomerases. Inactivation of DNA topoisomerase II does not alter significantly the time dependence of the patterns of nascent DNA chain synthesis, which is consistent with the notion that the requirement of this enzyme for viability is due to its essential role during mitosis, when pairs of intertwined newly replicated chromosomes are being segregated. The absence of DNA topoisomerase I leads to a temporary delay in the extension of the short DNA chains; this delay in chain elongation is also reflected in the rate of total DNA synthesis in the delta top1 mutant during the early S-phase. Thus, in wild-type cells, DNA topoisomerase I is probably the major replication swivel. The patterns of DNA synthesis in asynchronously grown delta top1 top2 ts cells at permissive and non-permissive temperatures are also consistent with the above conclusions.

DNA Replication↗
PubMed1989

All ras proteins are polyisoprenylated but only some are palmitoylated.

The C-terminal CAAX motif of the yeast mating factors is modified by proteolysis to remove the three terminal amino acids (-AAX) leaving a C-terminal cysteine residue that is polyisoprenylated and carboxyl-methylated. Here we show that all ras proteins are polyisoprenylated on their C-terminal cysteine (Cys186). Mutational analysis shows palmitoylation does not take place on Cys186 as previously thought but on cysteine residues contained in the hypervariable domain of some ras proteins. The major expressed form of c-K-ras (exon 4B) does not have a cysteine residue immediately upstream of Cys186 and is not palmitoylated. Polyisoprenylated but nonpalmitoylated H-ras proteins are biologically active and associate weakly with cell membranes. Palmitoylation increases the avidity of this binding and enhances their transforming activity. Polyisoprenylation is essential for biological activity as inhibiting the biosynthesis of polyisoprenoids abolishes membrane association of p21ras.

Cysteine↗
PubMed1993

Phenotypic effects in Saccharomyces cerevisiae after regulated expression of beta-(1,3)-glucanase from Nicotiana plumbaginifolia.

A recombinant yeast strain was constructed of which the cell wall porosity could be reversibly and conditionally modulated. This strain expresses the Nicotiana plumbaginifolia beta-(1,3)-glucanase under control of the Saccharomyces cerevisiae GAL1 promoter and the mating factor alpha 1 prepro-sequence. The following phenotypic effects were observed after expression of this enzyme: (a) expressed beta-(1,3)-glucanase is toxic to the producing yeast cells, which is reflected by a strong growth inhibition; as beta-(1,3)-glucanase could be detected only inside the cells, it seems to interfere with cell wall growth from within the cell; (b) after induction of glucanase, the recombinant strain lost up to 20% of some periplasmic enzymes, as evidenced by the release of normally periplasmic-associated invertase; (c) preliminary growth in a synthetic medium containing galactose significantly increased the transformation efficiency of the recombinant yeast strain.

Base Sequence↗
PubMed1994

Expression of human parathyroid hormone in mammalian cells, Escherichia coli and Saccharomyces cerevisiae.

The entire human parathyroid hormone (hPTH) cDNA gene with its natural signal and pro-region is expressed in transfected mouse mammary tumor cells (C127I cells) and Chinese hamster lung cells (DON cells) under control of the murine metallothioneine-1 promoter in a vector in which replication functions are provided by the entire genome of bovine papilloma virus type I (BPV-1). Authentic hPTH is efficiently produced by the non-endocrine cells and secreted to the growth medium without any abberant processing. Immunoblots from SDS-PAGE gels of concentrated growth medium reveal one band corresponding to intact, undegraded hPTH. Purification by reversed-phase HPLC results in a peptide with an amino acid content and N-terminal sequence identical to hPTH. For comparison, hPTH cDNA with deleted prepro-region is also expressed as secretory proteins in Escherichia coli and in Saccharomyces cerevisiae. In E. coli the vector construct is based on the staphylococcal protein A promoter employing protein A signal sequence. In S. cerevisiae a mating factor alpha expression system containing the factor alpha-signal sequence is employed. The results show that intact hPTH is secreted in addition to proteolytically cleaved fragments in both microorganisms. Thus, the signal sequences promote efficient secretion, and correct N-terminal processing of hPTH in both mammalian, bacterial and yeast cells. However, the folding characteristics of hPTH make it susceptible to internal proteolytical cleavage which appears to be species specific in yeast and E. coli.

Amino Acid Sequence↗
PubMed1987

Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast.

High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.

DNA, Recombinant↗
PubMed1989

The conformation of mature human alpha-amylase conditions its secretion from yeast.

The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.

Amino Acid Sequence↗
PubMed1989

Production and secretion of porcine urokinase in Saccharomyces cerevisiae: characterization of the secreted gene product.

The properties of porcine urokinase plasminogen activator (u-PA), produced and secreted by Saccharomyces cerevisiae, were studied to evaluate processing of the enzyme by yeast. Porcine u-PA cDNA was positioned behind the triosephosphate isomerase promoter and the yeast alpha-mating factor secretion signal sequences in a yeast expression vector, pZV125. Greater than 99% of the secreted PA activity was found to be single chain (pro-urokinase). The secreted gene product could be converted to two-chain (tc) with plasmin and then purified to homogeneity on benzamidine sepharose. Plasmin cleavage resulted in the formation of high Mr (HMW) and low Mr moieties representing HMW tc and free catalytic domain, respectively, as detected by N-terminal amino acid sequence analysis. Approximately 60-70% of the secreted activity was found to be associated with hyperglycosylated fractions from G-75 sizing columns. Approximately 30% of the total activity was secreted into the culture medium, where levels of activity approached 200 I.U./ml.

Animals↗
PubMed1993

Protein farnesyltransferase: production in Escherichia coli and immunoaffinity purification of the heterodimer from Saccharomyces cerevisiae.

Protein farnesylation in Saccharomyces cerevisiae is mediated by a heterodimeric enzyme, protein farnesyltransferase (PFTase), encoded by the genes RAM1 and RAM2. A series of plasmids for the expression of RAM1 and RAM2 in Escherichia coli was prepared and evaluated. Maximal production of functional PFTase was seen in strains containing a multicopy plasmid with a synthetic operon in which the RAM1 and RAM2 structural genes were translationally coupled by overlapping TAATG stop-start codons and by locating a ribosome-binding site near the 3' end of the upstream gene. This was accomplished by an insertional mutation at the 3'-end of RAM1 that embedded an AGGAGGAG sequence within codons for the tetrapeptide, QEEF, added to the end of the Ram1 protein. The QEEF C-terminal motif in the Ram1 subunit of PFTase facilitated purification of the enzyme by immunoaffinity chromatography on an anti-alpha-tubulin column prepared using monoclonal antibodies that recognized a tripeptide EEF epitope. Heterodimeric recombinant yeast PFTase::QEEF (re-PFTase::QEEF) constituted approximately 4% of total soluble protein in induced cells and was readily purified 25-fold in two steps by ion exchange and immunoaffinity chromatography in an overall 25% yield. Michaelis constants for farnesyl diphosphate (FPP) and Hras protein (modified to contain a yeast a-mating factor PACVIA sequence at the C terminus) were 5.5 and 15 microM, respectively; the kcat was 0.7 s-1.

Alkyl and Aryl Transferases↗