Expression and functional characterization of the mammalian intestinal peptide transporter PepT1 in the methylotropic yeast Pichia pastoris.
AuthorsDoring F, Theis S, Daniel H
JournalBiochem Biophys Res Commun
PubMed ID9126331
The methylotrophic yeast Pichia pastoris was used for heterologous expression of the rabbit intestinal peptide transporter PepT1 and its functional characterization. PepT1 mediates the electrogenic transmembrane transport of di- and tripeptides and peptido-mimetics such as beta-lactam antibiotics and ACE-inhibitors. Functional expression of PepT1 was determined in different recombinant clones by ... More
Recombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera.
AuthorsHarmsen MC, Heeringa P, van der Geld YM, Huitema MG, Klimp A, Tiran A, Kallenberg CG
JournalClin Exp Immunol
PubMed ID9367410
'The open reading frame of human proteinase 3 (PR3) without the prepro- peptide was cloned and expressed in Escherichia coli (rcPR3) and in Pichia pastoris (rpPR3). The 6-histidine tagged rpPR3 was efficiently secreted into culture supernatant from which it could be purified by immobilized metal chelate chromatography. Purified rpPR3 migrated ... More
Autocatalytic processing of recombinant human procathepsin L. Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro.
AuthorsMenard R, Carmona E, Takebe S, Dufour E, Plouffe C, Mason P, Mort JS
JournalJ Biol Chem
PubMed ID9468501
'The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent ... More
Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes.
AuthorsTanaka M, Suda T, Yatomi T, Nakamura N, Nagata S
JournalJ Immunol
PubMed ID9036978
'Fas ligand (FasL) is a type II membrane protein. Binding of FasL to its receptor, Fas, induces apoptosis. Matrix metalloproteinase cleaves the membrane-bound human FasL to yield the active soluble form. Here, we have produced a large amount of human soluble rFasL using the yeast, Pichia pastoris. The purified rFasL ... More
Role of the cysteine residues in the alpha1,2-mannosidase involved in N- glycan biosynthesis in Saccharomyces cerevisiae. The conserved Cys340 and Cys385 residues form an essential disulfide bond.
AuthorsLipari F, Herscovics A
JournalJ Biol Chem
PubMed ID8910350
'The Saccharomyces cerevisiae alpha1,2-mannosidase, which removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2, is a member of a family of alpha1,2-mannosidases with similar amino acid sequences. The yeast alpha1,2-mannosidase contains five cysteine residues, three of which are conserved. Recombinant yeast alpha1, 2- mannosidase, produced as the soluble catalytic ... More
Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway.
AuthorsSahu A, Isaacs SN, Soulika AM, Lambris JD
JournalJ Immunol
PubMed ID9605165
'Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a ... More
Expression of biologically active human vascular endothelial growth factor in yeast.
AuthorsMohanraj D, Olson T, Ramakrishnan S
JournalGrowth Factors
PubMed ID8527160
'Vascular endothelial growth factor (VEGF) is a glycoprotein consisting of two identical polypeptide chains linked by a disulfide bond. The unique biological activities of VEGF include its potent mitogenic and permeability inducing properties specific for the vascular endothelium. VEGF is implicated in tumor angiogenesis, wound healing, and the stimulation of ... More
Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain [published erratum appears in Protein Expr Purif 1997 Dec;11(3):304]
AuthorsHeimo H, Palmu K, Suominen I
JournalProtein Expr Purif
PubMed ID9179293
'In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc). Pichia pastoris produced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA from Aspergillus ... More
Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.
AuthorsLima CD, Klein MG, Weinstein IB, Hendrickson WA
JournalProc Natl Acad Sci U S A
PubMed ID8643579
'The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast ... More
Comparison of natural and recombinant isoforms of grass pollen allergens.
AuthorsPetersen A, Grobe K, Lindner B, Schlaak M, Becker WM
JournalElectrophoresis
PubMed ID9194614
'More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2- DE blotting using patients'' IgE and ... More
Cleavage-secretion of angiotensin I-converting enzyme in yeast.
AuthorsWilliams TA, Gouttaya M, Tougard C, Michaud A, Chauvet MT, Corvol P
JournalMol Cell Endocrinol
PubMed ID9140074
'Angiotensin I-converting enzyme (ACE) is a type I transmembrane protein composed of two domains (N and C domains) which undergoes a post- translational proteolytic cleavage in mammalian cells to release the soluble ectodomain. The protease involved in ACE cleavage-secretion (ACE-secretase) is not well characterised and eludes isolation: the presence of ... More
Expression of a human serum albumin variant with high affinity for thyroxine.
AuthorsPetersen CE, Ha CE, Mandel M, Bhagavan NV
JournalBiochem Biophys Res Commun
PubMed ID7575519
'In this study a protein expression system was used to synthesize recombinant human serum albumin containing a mutation that has been shown to result in familial dysalbuminemic hyperthyroxinemia. Equilibrium dialysis was used to measure the binding of this recombinant human serum albumin with thyroxine. The association constant determined for the ... More
Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.
AuthorsMistry AR, Falciola L, Monaco L, Tagliabue R, Acerbis G, Knight A, Harbottle RP, Soria M, Bianchi ME, Coutelle C, Hart SL
JournalBiotechniques
PubMed ID9105624
'This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native ... More
Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family.
AuthorsWuebbens MW, Roush ED, Decastro CM, Fierke CA
JournalBiochemistry
PubMed ID9100029
'The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino- terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four ... More
Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants.
AuthorsMcGrew JT, Leiske D, Dell B, Klinke R, Krasts D, Wee SF, Abbott N, Armitage R, Harrington K
JournalGene
PubMed ID9099880
'Pichia pastoris is a yeast capable of expressing large amounts of some proteins. When expression vectors are introduced into P. pastoris, individual transformants typically express widely varying amounts of protein. Because clones expressing the highest level of protein occur infrequently during the transformation process, finding them can be very labor-intensive. ... More
Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase.
AuthorsMartinet W, Saelens X, Deroo T, Neirynck S, Contreras R, Min Jou W, Fiers W
JournalEur J Biochem
PubMed ID9249044
'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 ... More
Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles.
AuthorsSugrue RJ, Fu J, Howe J, Chan YC
JournalJ Gen Virol
PubMed ID9266980
'We have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunoprecipitation, which is the ... More
Altered antigenicity of 'a' determinant variants of hepatitis B virus.
AuthorsChiou HL, Lee TS, Kuo J, Mau YC, Ho MS
JournalJ Gen Virol
PubMed ID9349486
'The ''a'' determinant of hepatitis B virus (HBV) surface antigen (HBsAg) is the most important target for diagnosis and immunoprophylaxis. Several HBV variants with point mutations within the ''a'' determinant have been identified among fully vaccinated children in Taiwan. We investigated the effect of each of these mutations on the ... More
Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris.
AuthorsFitzGerald K, Holliger P, Winter G,
JournalProtein Eng
PubMed ID9488147
'Diabodies are dimeric antibody fragments held together by associated heavy and light chain variable domains present on different polypeptide chains. To improve their stability we have introduced cysteine residues into the V-domains to promote the disulphide crosslinking of the dimer. A crosslinked bivalent diabody against carcinoembryonic antigen (CEA) and a ... More
Recombinant caspase-3 expressed in Pichia pastoris is fully activated and kinetically indistinguishable from the native enzyme.
AuthorsSun J, Bottomley SP, Kumar S, Bird PI
JournalBiochem Biophys Res Commun
PubMed ID9325193
'Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis. Recombinant caspases are typically produced in Escherichia coli expression systems with the attendant problems of solubilization, re-folding and activation of the protease. Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeast Pichia pastoris, and the ... More
Expression of biologically active beta subunit of bovine follicle- stimulating hormone in the methylotrophic yeast Pichia pastoris.
AuthorsSamaddar M, Catterall JF, Dighe RR
JournalProtein Expr Purif
PubMed ID9268682
'Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an alpha subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone- specific beta subunit. The objective of the present study is to develop a recombinant DNA expression system for the beta subunit of FSH ... More
High-level expression of recombinant Aplysia ADP-ribosyl cyclase in offhia pastoris by fermentation.
AuthorsMunshi C, Lee HC
JournalProtein Expr Purif
PubMed ID9325145
'Cyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD+, is rapidly emerging as an endogenous modulator of Ca2(+)- induced Ca2+ release mechanisms in various cellular systems. ADP ribosyl cyclase, first isolated from the marine invertebrate Aplysia californica, cyclizes NAD+ to cADPR. In this study we have utilized the ... More
Large-scale production of human CD38 in yeast by fermentation.
AuthorsMunshi CB, Fryxell KB, Lee HC, Branton WD
JournalMethods Enzymol
PubMed ID9211327
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Intracellular expression and purification of the c-kit receptor kinase domain in Pichia pastoris.
AuthorsLam LP, Berger SA
JournalBiotechniques
PubMed ID9232234
No abstract available.
Large-scale production in Pichia pastoris of the recombinant vaccine Gavac against cattle tick.
AuthorsCanales M, Enriquez A, Ramos E, Cabrera D, Dandie H, Soto A, Falcon V, Rodriguez M, de la Fuente J
JournalVaccine
PubMed ID9141213
A gene coding for the Bm86 tick protein was recently cloned, expressed in Pichia pastoris and shown to induce an inmunological response in cattle against ticks. Moreover, the Gavac vaccine (Heber Biotec S.A., Havana, Cuba), which contains this recombinant protein, has proved to control the Boophilus microplus populations under field ... More
Expression and analysis of heparin-binding regions of the amyloid precursor protein of Alzheimer's disease.
Deletion mutagenesis studies have suggested that there are two domains within APP which bind heparan sulphate. These domains have been cloned and expressed in the yeast Pichia pastoris. Both recombinant proteins bound to heparin. One domain (APP316-447) was further characterised by binding studies with peptides encompassing this region. Peptides homologous ... More
Construction and expression in the yeast Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-1 and B7-2 and the B7 counter-receptor CTLA-4.
AuthorsGerstmayer B, Pessara U, Wels W
JournalFEBS Lett
PubMed ID9141482
We have generated soluble recombinant forms of the costimulatory molecules B7-1 and B7-2, and their counter-receptor CTLA-4 using a yeast Pichia pastoris expression system. Fragments comprising the extracellular domains of human B7-1, B7-2, and CTLA-4 molecules were expressed at high levels and could be purified from culture supernatants following a ... More
Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex.
AuthorsLinnevers CJ, McGrath ME, Armstrong R, Mistry FR, Barnes MG, Klaus JL, Palmer JT, Katz BA, Bromme D
JournalProtein Sci
PubMed ID9098904
Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site. ... More
Structure and interactions of NCAM modules 1 and 2, basic elements in neural cell adhesion.
AuthorsJensen PH, Soroka V, Thomsen NK, Ralets I, Berezin V, Bock E, Poulsen FM
JournalNature Structural Biology
PubMed ID10331878
The structure in solution of the second Ig-module fragment of residues 117-208 of NCAM has been determined. Like the first Ig-module of residues 20-116, it belongs to the I set of the immunogloblin superfamily. Module 1 and module 2 interact weakly, and the binding sites of this interaction have been ... More
The first immunoglobulin-like neural cell adhesion molecule (NCAM) domain is involved in double-reciprocal interaction with the second immunoglobulin-like NCAM domain and in heparin binding.
AuthorsKiselyov VV, Berezin V, Maar TE, Soroka V, Edvardsen K, Schousboe A, Bock E
JournalJ Biol Chem
PubMed ID9092558
To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM domains ... More
Expression and characterization of the hepatitis E virus ORF3 protein in the methylotrophic yeast, Pichia pastoris.
AuthorsLal SK, Tulasiram P, Jameel S
JournalGene
PubMed ID9185850
We have used the methylotrophic yeast, Pichia pastoris, to express the open reading frame 3 (ORF3) of the hepatitis E virus (HEV). The ORF3 gene codes for a 123-amino-acid protein that contains highly immunodominant epitopes and is a potentially useful diagnostic and immunoprophylactic antigen. The expressed protein showed positive on ... More
Production of active polyomavirus large T antigen in yeast Pichia pastoris.
AuthorsPeng YC, Acheson NH
JournalVirus Res
PubMed ID9178495
The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2. Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by ... More
Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis -- a review
AuthorsGellissen G, Hollenberg CP
JournalGene
PubMed ID9185853
From the onset of gene technology yeasts have been among the most commonly used host cells for the production of heterologous proteins. At the beginning of this new development the attention in molecular biology and biotechnology focused on the use of the best characterized species, Saccharomyces cerevisiae, leading to an ... More
pPIC9-Fc: a vector system for the production of single-chain Fv-Fc fusions in Pichia pastoris as detection reagents in vitro.
AuthorsLiu J, Wei D, Qian F, Zhou Y, Wang J, Ma Y, Han Z,
JournalJ Biochem (Tokyo)
PubMed ID14769881
Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, there is no such universal system available up to now. A vector system was constructed based on pPIC9- Fc, in which the hinge, ... More
The role of electrostatic interactions in human serum albumin binding and stabilization by halothane.
Electrostatic interactions have been proposed as a potentially important force for anesthetics and protein binding but have not yet been tested directly. In the present study, we used wild-type human serum albumin (HSA) and specific site-directed mutants as a native protein model to investigate the role of electrostatic interactions in ... More
Structure of the C2 domain of human factor VIII at 1.5 A resolution.
Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs ... More
The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages.
AuthorsBorg-von Zepelin M, Beggah S, Boggian K, Sanglard D, Monod M
JournalMol Microbiol
PubMed ID9632257
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 ... More
Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris.
AuthorsMorel N, Massoulie J
JournalBiochem J
PubMed ID9359842
In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETDelta, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEDelta, reduced to the catalytic domain). ... More
High-level production of recombinant Geotrichum candidum lipases in yeast Pichia pastoris.
AuthorsHolmquist M, Tessier DC, Cygler M
JournalProtein Expr Purif
PubMed ID9325136
We describe the heterologous high-level expression of the two Geotrichum candidum lipase (GCL) isoenzymes from strain ATCC 34614 in the methylotrophic yeast Pichia pastoris. The lipase cDNAs were placed under the control of the methanol-inducible alcohol oxidase promoter. The lipases expressed in P. pastoris were fused to the alpha-factor secretion ... More
High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.
AuthorsHasslacher M, Schall M, Hayn M, Bona R, Rumbold K, Luckl J, Griengl H, Kohlwein SD, Schwab H
JournalProtein Expr Purif
PubMed ID9325140
(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in ... More
Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.
AuthorsJerva LF, Sullivan G, Lolis E
JournalProtein Sci
PubMed ID9260277
Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha- chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP- 2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, ... More
Effect of chromophore exchange on the resonance Raman spectra of recombinant phytochromes.
AuthorsKneip C, Mozley D, Hildebrandt P, Gartner W, Braslavsky SE, Schaffner K
JournalFEBS Lett
PubMed ID9305725
The recombinant 65-kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (PphiB) and phycocyanobilin (PCB), respectively. The Pr and Pfr states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA ... More
Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus.
AuthorsCalera JA, Paris S, Monod M, Hamilton AJ, Debeaupuis JP, Diaquin M, Lopez-Medrano R, Leal F, Latge JP
JournalInfect Immun
PubMed ID9353056
Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding ... More
Evidence for the formation of a heterotrimeric complex of leukaemia inhibitory factor with its receptor subunits in solution.
AuthorsZhang JG, Owczarek CM, Ward LD, Howlett GJ, Fabri LJ, Roberts BA, Nicola NA
JournalBiochem J
PubMed ID9271090
Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high-affinity, functional, receptor complex. Human LIF binds with unusually high affinity to a naturally occurring mouse soluble LIF receptor alpha- chain, ... More
Cloning, expression, purification, and characterization of the murine lysosomal acid alpha-mannosidase.
AuthorsMerkle RK, Zhang Y, Ruest PJ, Lal A, Liao YF, Moremen KW
JournalBiochim Biophys Acta
PubMed ID9305783
Catabolism of alpha-linked mannose residues on eukaryotic glycoproteins is accomplished by a broad specificity lysosomal alpha-mannosidase (EC 3.2.1.24). Based on regions of protein sequence conservation between the lysosomal alpha-mannosidase from Dictyostelium discoideum and the murine Golgi glycoprotein processing alpha 1,3/1,6-mannosidase, alpha- mannosidase II, we have cloned a cDNA encoding the ... More
Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans
AuthorsBeauvais A, Monod M, Wyniger J, Debeaupuis JP, Grouzmann E, Brakch N, Svab J, Hovanessian AG, Latge JP
JournalInfect Immun
PubMed ID9234752
A dipeptidyl-peptidase IV was purified from the culture medium of the human-pathogenic fungus Aspergillus fumigatus. The enzyme has an apparent molecular mass of 95 kDa and contained approximately 10 kDa of N-linked carbohydrate. This glycoprotein is antigenic and has all characteristics of the class IV dipeptidyl-peptidases: removal of Xaa-Pro and ... More
Expression of human amyloid precursor protein ectodomains in Pichia pastoris: analysis of culture conditions, purification, and characterization.
AuthorsHenry A, Masters CL, Beyreuther K, Cappai R
JournalProtein Expr Purif
PubMed ID9226725
We have examined the use of the yeast Pichia pastoris for expression of the human amyloid precursor protein (APP). The ectodomains of the isoforms APP695, APP751, and APP770 were expressed in both P. pastoris protease-deficient strain SMD1163 and wild-type strain GS115, using the secretion vector pHIL-S1. Expression of recombinant APP ... More
Costimulation of T cell proliferation by a chimeric B7-2 antibody fusion protein specifically targeted to cells expressing the erbB2 proto-oncogene.
AuthorsGerstmayer B, Altenschmidt U, Hoffmann M, Wels W
JournalJ Immunol
PubMed ID9144470
T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with antigenic peptides in the context of MHC molecules. The second, costimulatory signal can be provided by cell surface molecules on APCs such as ... More
Role of tryptophan-63 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability, folding, and ligand binding properties.
AuthorsChang Y, Zajicek J, Castellino FJ
JournalBiochemistry
PubMed ID9201906
Conservative (F and Y) and radical (H and S) mutations have been engineered at a rigidly conserved aromatic residue, W63, of the isolated recombinant kringle 2 domain of tissue-type plasminogen activator (r-K2tPA), an amino acid residue predicted from the X-ray crystal structure to be important in the ligand binding properties ... More
Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris.
AuthorsAbdulaev NG, Popp MP, Smith WC, Ridge KD
JournalProtein Expr Purif
PubMed ID9179292
The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal. Quantitative-competitive PCR analysis of ... More
The Golgi sialoglycoprotein MG160, expressed in Pichia pastoris, does not require complex carbohydrates and sialic acid for secretion and basic fibroblast growth factor binding.
AuthorsChen YJ, Gonatas NK
JournalBiochem Biophys Res Commun
PubMed ID9168962
MG160, a type I membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, shows high homology (over 90%) with CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand of the cell surface of murine myeloid cells. When Chinese Hamster Ovary (CHO) cells were stably transfected with ... More
Characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambda1 protein.
AuthorsBisaillon M, Bergeron J, Lemay G
JournalJ Biol Chem
PubMed ID9218469
Previous studies have shown that the reovirus lambda1 core protein harbors a putative nucleotide-binding motif and exhibits an affinity for nucleic acids. In addition, a nucleoside triphosphate phosphohydrolase activity present in reovirus cores has been recently assigned to lambda1 using gene reassortment analysis. In this study, it was demonstrated that ... More
Production of the rat complement regulator, Crry, as an active soluble protein in Pichia pastoris.
AuthorsHe C, Alexander JJ, Lim A, Quigg RJ
JournalArch Biochem Biophys
PubMed ID9169025
In this report, we describe the use of the methylotrophic yeast Pichia pastoris for the production of the rat complement regulator, Crry. Crry normally exists as an intrinsic membrane protein containing six to seven short consensus repeats (SCRs), a transmembrane region, and a cytoplasmic tail. To produce Crry as a ... More
Ktr1p is an alpha-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris.
AuthorsRomero PA, Lussier M, Sdicu AM, Bussey H, Herscovics A
JournalBiochem J
PubMed ID9020857
The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the alpha 1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to ... More
Expression and secretion of rabbit plasma cholesteryl ester transfer protein by Pichia pastoris.
AuthorsKotake H, Li Q, Ohnishi T, Ko KW, Agellon LB, Yokoyama S
JournalJ Lipid Res
PubMed ID8728322
The rabbit cholesteryl ester transfer protein (CETP) was expressed in the methylotrophic yeast Pichia pastoris by introducing the CETP cDNA under the control of the methanol-inducible alcohol oxidase promoter. The cDNA was cloned from in vitro amplified cDNA of rabbit liver mRNA. The nucleotide sequence of the cloned cDNA differed ... More
Hexose oxidase from the red alga Chondrus crispus. Purification, molecular cloning, and expression in Pichia pastoris.
AuthorsHansen OC, Stougaard P
JournalJ Biol Chem
PubMed ID9111074
Hexose oxidase from Chondrus crispus catalyzes the oxidation of a variety of mono- and disaccharides including D-glucose, D-galactose, maltose, and lactose. The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein with a relative molecular mass of approximately 130,000 (Sullivan, J. D., and ... More
Overexpression and characterization of Aspergillus awamori wild-type and mutant glucoamylase secreted by the methylotrophic yeast Pichia pastoris: comparison with wild-type recombinant glucoamylase produced using Saccharomyces cerevisiae and Aspergillus niger as hosts.
AuthorsFierobe HP, Mirgorodskaya E, Frandsen TP, Roepstorff P, Svensson B
JournalProtein Expr Purif
PubMed ID9056481
Glucoamylase from Aspergillus niger (identical to Aspergillus awamori glucoamylase) is an industrially important, multidomain N- and O- glycosylated starch-hydrolase. To produce protein-engineered glucoamylase, heterologous expression is established in the methylotrophic yeast Pichia pastoris. Using the vector pHIL-D2, the cDNA encoding A. awamori glucoamylase is inserted in the yeast genome downstream ... More
Role of the occluding loop in cathepsin B activity.
AuthorsIlly C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS
JournalJ Biol Chem
PubMed ID8995421
Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of ... More
Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.
AuthorsMonosov EZ, Wenzel TJ, Luers GH, Heyman JA, Subramani S
JournalJ Histochem Cytochem
PubMed ID8666743
We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia ... More
Influence of expression system on chromophore binding and preservation of spectral properties in recombinant phytochrome A.
AuthorsGartner W, Hill C, Worm K, Braslavsky SE, Schaffner K
JournalEur J Biochem
PubMed ID8665921
N-Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C-terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation. The start and end positions of the cDNA employed (phyA from oat) mimic the ... More
Identification of residues in the first domain of human Fc alpha receptor essential for interaction with IgA.
The FcR family contains multiple receptors for Igs, of which the most distantly related ( approximately 20%) is the IgA receptor (human Fc alpha R), being more homologous ( approximately 35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the Fc ... More
Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.
AuthorsBird CH, Sutton VR, Sun J, Hirst CE, Novak A, Kumar S, Trapani JA, Bird PI
JournalMol Cell Biol
PubMed ID9774654
Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect ... More
Mutations in a specific human serum albumin thyroxine binding site define the structural basis of familial dysalbuminemic hyperthyroxinemia.
AuthorsPetersen CE, Ha CE, Jameson DM, Bhagavan NV
JournalJ Biol Chem
PubMed ID8702585
The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and ... More
A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer.
AuthorsSim BK, O'Reilly MS, Liang H, Fortier AH, He W, Madsen JW, Lapcevich R, Nacy CA
JournalCancer Res
PubMed ID9102221
Endogenous murine angiostatin, identified as an internal fragment of plasminogen, blocks neovascularization and growth of experimental primary and metastatic tumors in vivo. A recombinant protein comprising kringles 1-4 of human plasminogen (amino acids 93-470) expressed in Pichia pastoris had physical properties (molecular size, binding to lysine, reactivity with antibody to ... More
Identification and characterization of two alternative splice variants of human interleukin-2.
AuthorsTsytsikov VN, Yurovsky VV, Atamas SP, Alms WJ, White B
JournalJ Biol Chem
PubMed ID8798495
Our previous work showed that alternative splicing is used to make an inhibitory variant of human interleukin (IL)-4. Because of homology between IL-4 and IL-2 proteins and receptors, we tested whether alternative splicing is used to generate similar inhibitory variants of human IL-2. Messenger RNA from peripheral blood mononuclear cells ... More
ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium- mobilizing metabolite from NADP.
AuthorsAarhus R, Graeff RM, Dickey DM, Walseth TF, Lee HC
JournalJ Biol Chem
PubMed ID8530456
ADP-ribosyl cyclase catalyzes the cyclization of NAD+ to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca(2+)-induced Ca2+ release mechanism in cells. CD38 is a lymphocyte differentiation antigen which has recently been shown to be a bifunctional enzyme that can synthesize cADPR from NAD+ as ... More
Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris.
AuthorsJonsson LJ, Saloheimo M, Penttila M
JournalCurr Genet
PubMed ID9388299
A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia ... More
Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer.
Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, alpha-galactosidase A (alpha-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the alpha-gal A ... More