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Showing papers on "Pichia pastoris published in 1998"


Journal ArticleDOI
TL;DR: These studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of per oxisome homeostasis.
Abstract: We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

246 citations


Book ChapterDOI
TL;DR: This Applications Report presents a simple protocol for achieving high-density culture of Pichia pastoris (P. pastoris) cells using a New Brunswick benchtop, autoclavable stirred-tank fermentor or bioreactor.
Abstract: This Applications Report presents a simple protocol for achieving high-density culture of Pichia pastoris (P. pastoris) cells using a New Brunswick benchtop, autoclavable stirred-tank fermentor or bioreactor. Introduction to Pichia Pastoris in a Stirred-Tank Fermentor Richard Mirro, Eppendorf Inc., Enfield, CT, U.S.A.

215 citations


Journal ArticleDOI
TL;DR: A role for Sap4 to Sap6 in pathogenicity is supported, as a high production of Sap4, Sap5 and Sap6 by C. albicans cells after phagocytosis by murine peritoneal macrophages is demonstrated.
Abstract: Medically important yeasts of the genus Candida secrete aspartyl proteinases (Sap), which are of particular interest as virulence factors. Six closely related gene sequences, SAP1 to SAP6, for secreted proteinases are present in Candida albicans. The methylotrophic yeast Pichia pastoris was chosen as an expression system for preparing substantial amounts of each Sap isoenzyme. Interestingly, Sap4, Sap5 and Sap6, which have not yet been detected in C. albicans cultures in vitro, were produced as active recombinant enzymes. Different Sap polyclonal antibodies were raised in rabbits and tested before further application by enzyme-linked immunosorbent assay (ELISA) against each recombinant Sap. Two antisera recognized only Sap4 to Sap6. Using these antisera, together with sap null mutants obtained by targeted mutagenesis, we could demonstrate a high production of Sap4, Sap5 and Sap6 by C. albicans cells after phagocytosis by murine peritoneal macrophages. Furthermore, a delta sap4,5,6 null mutant was killed 53% more effectively after contact with macrophages than the wild-type strain. These results support a role for Sap4 to Sap6 in pathogenicity.

182 citations


Journal ArticleDOI
15 Jun 1998-Yeast
TL;DR: A set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris are described.
Abstract: The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial beta-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF0279959; pIB3, AF027960; pIB4, AF027961.

177 citations


Journal ArticleDOI
17 Aug 1998-Gene
TL;DR: It is shown that the FLD1 promoter (PFLD1) is strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source, allowing the investigator a choice of carbon or nitrogen source (methylamine) regulation with the same expression strain.

163 citations


Journal ArticleDOI
TL;DR: It is concluded that the ability of endostatin to bind Zn2+ is essential for its antiangiogenic activity.

137 citations


Journal ArticleDOI
TL;DR: A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system and the structure confirms the proposed existence of five disulfide bonds.
Abstract: A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system. The antifreeze activity of recombinant SRAFP is indistinguishable from that of the wild-type protein. The global fold of SRAFP has been determined by two-dimensional 1H homonuclear and three-dimensional 1H-?15N? heteronuclear NMR spectroscopy using 785 NOE distance restraints and 47 angular restraints. The molecule folds into one globular domain that consists of two helices and nine beta-strands in two beta-sheets. The structure confirms the proposed existence of five disulfide bonds. The global fold of SRAFP is homologous to C-type lectins and pancreatic stone proteins, even though the sequence identity is only approximately 20%.

125 citations


Journal ArticleDOI
TL;DR: A simple and inexpensive methanol control system will help bioengineering studies on the production of recombinant proteins in P. pastoris, the growth and production of objective proteins in which the energy for the production competed with that for cell growth.

125 citations


Journal ArticleDOI
TL;DR: From these results, it is apparent that most foreign proteins secreted from P. pastoris are not subjected to the extensive mannosylation (hyperglycosylation) that commonly occurs in proteinssecreted from S. cerevisiae.

113 citations


Journal ArticleDOI
TL;DR: The data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins, and suggests that the two NB sites cannot function independently as catalytic sites in the intact molecule.
Abstract: Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

111 citations


Journal ArticleDOI
TL;DR: The current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed and the advantages and limitations of these systems are discussed in relation to S. cerevisiae.
Abstract: Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.

Journal ArticleDOI
TL;DR: Two distinct antigens derived from the dermatophyte Trichophyton that serve as targets for diverse immune responses in humans are defined.

Journal ArticleDOI
TL;DR: Various methods are used to demonstrate that Px7p is both cytosolic and intraperoxisomal, suggesting that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes.
Abstract: Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2) Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1 Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata The corresponding PpPex7p mutant proteins were stably expressed in P pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence

Journal ArticleDOI
TL;DR: IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins.

Journal ArticleDOI
TL;DR: It is demonstrated that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor.
Abstract: Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae alpha-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the beta2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the alpha-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the beta2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of beta2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems. Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence. Here, the Kex2 endopeptidase failed to process the alpha-factor leader correctly. Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing. In contrast, the beta2-adrenergic receptor fusion to the alpha-factor leader was correctly processed by the internal Kex2 endopeptidase. The Kex2-processed beta2-adrenergic receptor was not glycosylated. In conclusion, the high-level production of the two receptors in P. pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.

Journal ArticleDOI
TL;DR: A recombinant Hc fragment of botulinum neurotoxin, serotype B (rBoNTB(Hc), has been successfully expressed in a Mut+ strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine.

Journal ArticleDOI
TL;DR: Results show that short O‐linked saccharides of mannose containing (α1‐2) glycosidic linkages are present in P. pastoris cells and expressed proteins.
Abstract: O-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan-protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (alpha1-2)-linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1-4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides. Only a very small quantity of N-linked oligosaccharides was present on the Asn289 consensus site. The major products were O-linked (alpha1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O-linked saccharides of mannose containing (alpha1-2) glycosidic linkages are present in P. pastoris cells and expressed proteins.

Journal ArticleDOI
15 Jan 1998-Yeast
TL;DR: A protein expression system in the methylotrophic yeast, Pichia methanolica, is described and methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild‐type ADE2 gene.
Abstract: We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0.5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity.

Journal ArticleDOI
TL;DR: Recombinant ROL lipase was purified to homogeneity by a simple two-step purification procedure and had a specific activity of 8571 U mg-1 (triolein, 30 degrees C, pH 8.1) which is comparable with the purified native enzyme.

Journal ArticleDOI
TL;DR: The results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins.
Abstract: IgE-mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years. We purified natural Hev b 7, a 43-kDa patatin-like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences. A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library. The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa. The deduced amino acid sequence had 39−42 % identity to patatins from Solanum tuberosum. The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S. tuberosum, esterase activity. Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex-sensitized allergic individuals. In contrast to patatins from S. tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N-terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated. These results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins.

Journal ArticleDOI
TL;DR: Inhibitors found to be effective towards the recombinant 11β‐hydroxysteroid dehydrogenase include synthetic steroids and xenobiotic compounds, revealing selective inhibition of the reaction direction, useful for development of specific inhibitors.

Journal ArticleDOI
15 Mar 1998-Yeast
TL;DR: The alpha 1-antitrypsin (α 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues as mentioned in this paper.
Abstract: Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

Journal ArticleDOI
TL;DR: Recombinant procarboxypeptidase A2 was purified to homogeneity, and it was shown that its mature active form displays functional properties similar to those of the enzyme directly isolated from human pancreas.

Journal ArticleDOI
TL;DR: The cDNA encoding sucrose‐fructan 6‐Fructosyltransferase from barley has been expressed in the methylotrophic yeast Pichia pastoris using a translational fusion into vector pPICZαC, containing the N‐terminal signal sequence of Saccharomyces cerevisiae α‐factor to allow entry into the secretory pathway.

Journal ArticleDOI
TL;DR: The results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.
Abstract: The koji mold Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an A. oryzae library by using the A. fumigatus dppIV gene as a probe. Reverse transcriptase PCR experiments showed that the A. oryzae dppIV gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into A. oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The dppIV null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector Pichia pastoris. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.

Journal ArticleDOI
TL;DR: Batch fermentations were used to study the effect of different glycerol concentrations and pH conditions on growth of recombinantPichia pastoris, and there were no differences between Mut+ and Mut- strains during cell growth on Glycerol.
Abstract: Batch fermentations were used to study the effect of different glycerol concentrations and pH conditions on growth of recombinantPichia pastoris. Two strains ofP. pastoris were used: a wild-type in methanol utilization (Mut+) and a mutant defective in methanol utilization (Mut-). Under constant pH conditions of 5.0, glycerol concentrations up to 12% were efficiently utilized. Cell yield (Yx/s) of about 0.8 and a final cell density of about 95 g/L (dry cell) were achieved. However, there were significant differences (probability [Pr]> F 0.0351) in specific growth rates between the initial glycerol concentrations of 2, 7, and 12%. When fermentations were conducted without pH control, growth continued until the pH had decreased to about 2.5. Growth stopped at pH 2.2 with uncontrolled pH, and residual glycerol concentrations were greater than 2%. As a result, Yx/s decreased to about 0.3. There were no differences between Mut+ and Mut- strains during cell growth on glycerol.

Journal ArticleDOI
TL;DR: A simple and efficient purification procedure of a recombinant beta-(1-6)-glucanase from Trichoderma harzianum expressed in Pichia pastoris is described, which will facilitate studies of the molecular organization of the fungal cell wall.

Journal ArticleDOI
TL;DR: The recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals and for inducing protection of mice against a lethal challenge with T. gondii tachyzoites.

Journal ArticleDOI
TL;DR: Recombinant human pancreatic lipase was expressed in broth cultures and was purified from the medium by DEAE blue Sepharose and hydroxyapatite chromatography, resulting in a highly purified lipase that was inhibited by bile salts, required colipase for activity, and demonstrated interfacial activation.

Patent
31 Jul 1998
TL;DR: In this article, regulatory nucleotide sequences for a novel Pichia pastoris gene, designated PpSEC10 gene, and respective amino acid sequences for the secretion leader and mature Sec10p protein components of the precursor polypeptide encoded by this novel gene are provided.
Abstract: Regulatory nucleotide sequences for a novel Pichia pastoris gene, designated PpSEC10 gene, and the nucleotide sequences and respective amino acid sequences for the secretion leader and the mature Sec10p protein components of the precursor polypeptide encoded by this novel gene are provided. These compositions are useful in methods for expression and secretion of proteins when assembled in proper reading frame, individually or in combination, within a DNA construct that further comprises a nucleotide sequence encoding a protein of interest. Vectors comprising the DNA constructs of the invention can be used to transform a yeast host cell, which can then be cultured to obtain the secreted protein of interest. Kits useful in this method and in methods of detection of the Sec10p protein using antibodies are also disclosed.