SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
As a key part of the mechanism which controls growth and division, cells are able to respond to a variety of intracellular and extracellular stimuli. Significant progress has been made in the understanding of the biochemical mechanisms underlying mating-factor signal transduction in Saccharomyces cerevisiae. Some of these mechanisms may be relevant to the regulation of other signal transduction pathways.
Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris.
Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of alpha-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.
CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.
Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.
Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine was overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS-PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed.
Methods for the expression in Pichia pastoris and purification of the human activin receptor type I and II extracellular domains (ARIa/ARIb-ECDs, ARIIA/ARIIB-ECDs) are described. Key experimental aspects are also documented of the vector transformation methodology and the binding characteristics of these ECDs with activin A and inhibin. The cDNA constructs for these ECDs contained a C-terminal His6-tag with either the native signal (N) or the yeast alpha mating factor (alphaMF) sequence and were introduced into the pPICZ expression vector either as a single-copy or as a four-copy expression cassette. Hyper-resistant transformants (zeo(R): 500 microg/mL) generated from the cassette containing a single copy of the expression vector gave the stronger signal intensity with a DNA dot-blot screening assay. These transformants also produced higher quantities of the corresponding recombinant protein compared to transformants using the four-copy cassette vector. All receptor-ECD proteins expressed were found to be heterogeneously glycosylated, whereby the ARIIA-ECD and ARIIB-ECD had undergone two Asn-linked glycosylation events and the ARIb-ECD a single event. By SDS-PAGE, the de-glycosylated proteins migrated larger than the expected core size, indicating that they may have undergone O-linked glycosylation. Biacore-based procedures with the glycosylated and de-glycosylated ARIIA-ECD were employed to determine the kinetic and equilibrium binding parameters for the interaction with activin A and inhibin. The glycosylated ARIIA-ECD bound to activin A with a KD of 11.9 nM and inhibin with a KD of 21.1 nM. Although glycosylation of ARIIA-ECD was not strictly required for high affinity interactions with activin A or inhibin, it markedly improved the overall stability of the ARIIA-ECD.
The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae. The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity. The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha. The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S. cerevisiae.
A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.
It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule.
Rat mast cell protease 7 (rMCP7) is a neutral serine protease and a component of mast cells, where it is stored in secretory granules. Mast cells express numerous proteases so in order to characterize rMCP7, it was cloned and expressed as a recombinant protein in Pichia pastoris. During expression, rMCP7 protein was cleaved from the alpha-mating factor signal at the engineered KEX2 cleavage site to produce active rMCP7. The protein produced was stable at pH 5.5 and active in the absence of heparin. The rMCP7 was glycosylated and treatment with N-glycosidase F resulted in a protein of the predicted molecular mass of 30 kDa. The rMCP7 was purified via an ammonium sulfate precipitation, using casein as a carrier protein, followed by cation exchange chromatography. The purified protein was assayed using a range of substrates and where possible, k(m) and k(cat) values were determined. The substrate profile displayed by the recombinant rMCP7 was consistent with that of tryptase isolated from rat skin. The expression and purification of recombinant rMCP7 offer an efficient, low-cost method of producing large amounts of protein. It also offers the opportunity of easy manipulation and mutagenesis of rMCP7 for further biochemical, structural, and physiological studies.
Recombinant human midkine (rh-midkine) was efficiently produced in Pichia pastoris using the pre-pro secretion signal of yeast alpha-mating factor under the control of the AOX1 promoter. The pep4 host SMD1168 was used. The expression was induced at pH 3 and 20 degrees C in high cell-density fermentation and approximately 360 mg rh-midkine was secreted into 1L of medium. The authentic midkine could be obtained after one-step purification. Mass spectrometry of purified rh-midkine demonstrated a single large signal for the molecular ion [M + H](+) at 13241.2 m/z. This mass is identical to the authentic, unmodified human midkine. The precursor of rh-midkine was correctly processed in P. pastoris cells, yielding mature rh-midkine. Mass spectrometry detected no yeast-specific O-mannosylations in the purified midkine preparations. The circular dichroic spectrum indicated only a negative Cotton effect at 215 nm. Only beta-structures were indicated for the rh-midkine molecule in solution. Purified rh-midkine was active in a cell-proliferation assay.
Plant defensins are small cysteine-rich proteins that present high activity against fungi and bacteria and inhibition of insect proteases and alpha-amylases. Here, we present the expression in Pichia pastoris, purification and characterization of the recombinant Pisum sativum defensin 1(rPsd1); a pea defensin which presents four disulfide bridges and high antifungal activity. For this, we had to overcome the inefficiency of the STE13 protease. Our strategy was to clone the corresponding cDNA directly in-frame with a variant of the widely used secretion signal from the Saccharomyces cerevisiae alpha-mating factor, devoid of the STE13 proteolytic signal cleavage sequence. Using an optimized expression protocol, which included a buffered basal salt media formulation, it was possible to obtain about 63.0mg/L of 15N-labeled and unlabeled rPsd1. The recombinants were purified to homogeneity by gel filtration chromatography, followed by reversed-phase HPLC. Mass spectrometry of native and recombinant Psd1 revealed that the protein expressed heterologously was post-translationally processed to the same mature protein as the native one. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein had the same folding when compared to native Psd1. In addition, the rPsd1 was fully active against Aspergillus niger, if compared with native Psd1. To our knowledge, this is the first heterologous expression of a fully active plant defensin in a high-yield flask.
A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.
The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.
A yeast-like fungus Aureobasidium pullulans var. melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-1,4-beta-xylanase with an optimum pH of 2.0 [Ohta et al., J. Biosci. Bioeng., 92, 262-270 (2001)]. The xynI cDNA encoding the precursor protein (XynI) was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter. The 34 amino acid prepro-signal peptide of the A. pullulans XynI directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium. The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae alpha-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P. pastoris acid phosphatase. The N-terminal amino acid sequence and the apparent M(r) of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A. pullulans XynI signal sequence in P. pastoris. The three-dimensional model and mutational analysis of the xynI gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft, respectively, play a significant role in its low pH optimum.
Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post-translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, plasma membrane localization of Ras2p is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control. Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope. Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals. The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+). The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p. Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared. Four of the mutants, H194A, E156A, C251A, and H248A, were inactive. Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease.
Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H(2)O(2) oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.