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Results for “Heterologous expression system”

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PubMed2006

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.

Animals↗
PubMed2001

A region of the rat N-methyl-D-aspartate receptor 2A subunit that is sufficient for potentiation by phorbol esters.

N-methyl-D-aspartate (NMDA) receptors are modulated by protein kinase C (PKC) in vivo and in heterologous expression systems. In heterologous expression systems, PKC-mediated modulation is subunit specific with NR2A-containing receptors being potentiated by phorbol 12-myristate 13-acetate (PMA), while NR2C-containing receptors are inhibited or unaffected. In the present study we have produced chimeric receptors containing NR2A and NR2C to define the components of NR2A which are sufficient for potentiation by PMA. Amino acids 1105-1400 of NR2A placed onto the C-terminus of NR2C at amino acid 1102 was the minimum region sufficient for producing a PMA-stimulated receptor. This suggests that this region contains structural determinants for PKC-mediated potentiation of NR2A receptors.

Calcium↗
PubMed2003

Characterisation of mammalian GLUT glucose transporters in a heterologous yeast expression system.

We have developed a new heterologous expression system for mammalian glucose transporters. The system is based on a Saccharomyces cerevisiae strain completely deleted for all its endogenous hexose transporters and unable to take up and to grow on hexoses. To target the heterologous glucose transporters into the yeast plasma membrane in a fully active form, additional mutations had to be introduced into the hexose transport-deficient strain. Although GLUT1 was localized at the cell surface already in the parent strain, it supported uptake of glucose only in an deltaHXT FGY1-1 mutant strain. Moreover, various mutations within the first half of the second predicted transmembrane helix converted GLUT1 into a form able to support uptake of glucose into yeast cells. GLUT4 was trapped in intracellular structures but became functionally expressed in the plasma membrane in deltaHXT FGY1-1 FGY4X mutant strains. Glucose transport kinetics were determined with intact yeast cells by zero-trans influx measurements with a Km of 3.2 mM for human GLUT1 and of 12.6 mM for human GLUT4. Cytochalasin B inhibited these activities. Growth tests revealed that both transporter proteins are able to mediate uptake of glucose, mannose and galactose, but not of fructose. The new heterologous expression system should be a valuable tool to develop cell based high-throughput screening assays for identifying pharmaceutical compounds influencing the transporters.

Amino Acid Sequence↗
PubMed1996

Studies on the toxicity of analogues of dapsone in-vitro using rat, human and heterologously expressed metabolizing systems.

Three metabolizing systems (rat, heterologously expressed CYP3A4 and human liver) were used to evaluate 12 analogues of dapsone (4,4'diaminodiphenylsulphone) in-vitro. Methaemoglobin formation in a two-compartment and cytotoxicity in a single-compartment model were studied using human erythrocytes and neutrophils, respectively, as target cells. In the two-compartment system using rat microsomes as a generating system and methaemoglobin as an endpoint, the least potent methaemoglobin formers tested were the 2-methyl-4-propylamino (AXDD14), 2-hydroxy-4-4'amino (ABDD5) derivatives and a sulphone/trimethoprim derivative (K-130). Dapsone itself, a 2-methoxy-4-ethylamino (W10) and a 2-hydroxyl-4-ethylamino compound (ABDD39) were the most toxic. In the single-compartment cytotoxicity test using rat microsomes, AXDD14 was again among the least toxic, as was a 2-methyl 4-cyclopentyl derivative (AXDD17) and surprisingly ABDD39. The most cytotoxic compounds again included dapsone itself as well as two 2-trifluoromethyl derivatives. The only significant methaemoglobin formation and cytotoxicity shown with the heterologously expressed human CYP 3A4 was with AXDD14, which was extensively activated. Interestingly, metabolism of dapsone was low using the expressed CYP 3A4. In the two-compartment system using human liver microsomes, AXDD14, K-130 and ABDD5 were oxidized to a significantly lesser extent compared with dapsone and these preliminary findings indicate that future development of these compounds may be worthwhile.

Animals↗
PubMed1998

A system for heterologous expression of bacteriocins in Lactobacillus sake.

A system for efficient heterologous expression of class II bacteriocins is described that is based on introducing two plasmids in a bacteriocin-negative Lactobacillus sake strain. The first plasmid (pSAK20) contains the genes necessary for transcriptional activation of the Sakacin A promoter as well as export and processing of bacteriocin precursors. The second plasmid (a pLPV111 derivative) contains the structural and immunity genes for the bacteriocin of interest fused to the sakacin A promoter. Using this system, various bacteriocins were produced at levels equal to or higher than those obtained with the corresponding wild-type producer strains.

Bacteriocins↗
PubMed1999

Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems.

Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells. Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family. Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems. However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells. These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody. An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS-PAGE and hydroxyapatite chromatography. Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30-31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme.

Amino Acid Sequence↗
PubMed2005

Expression of plant cyclic nucleotide-gated cation channels in yeast.

The functional properties of inwardly conducting plant cyclic nucleotide-gated cation channels (CNGCs) have not been thoroughly characterized due in part to the recalcitrance of their functional expression in heterologous systems. Here, K+ uptake-deficient mutants of yeast (trk1,2) and Escherichia coli (LB650), as well as the Ca2+-uptake yeast mutant mid1,cch1, were used for functional characterization of Arabidopsis thaliana CNGCs, with the aim of identifying some of the cultural and physiological conditions that impact on plant CNGC function in heterologous systems. Use of the Ca2+-uptake yeast mutant provided the first evidence consistent with Ca2+ conduction by the A. thaliana CNGC AtCNGC1. Expression of AtCNGC1 in LB650 demonstrated that mutants of Escherichia coli (which has no endogenous calmodulin) can also be used to study functional properties of CNGCs. Expression of AtCNGC2 and AtCNGC4 enhanced growth of trk1,2 in the presence of hygromycin; AtCNGC1 has less of an effect. Deletion of the AtCNGC1 calmodulin-binding domain enhanced growth of trk1,2 at low external K+ but not of LB650, suggesting that yeast calmodulin may bind to, and down-regulate this plant channel. In vitro binding studies confirmed this physical interaction. Northern analysis, green fluorescent protein:AtCNGC1 fusion protein expression, as well as an antibody raised against a portion of AtCNGC1, were used to monitor expression of AtCNGC1 and deletion constructs of the channel in the heterologous systems. In the presence of the activating ligand cAMP, expression of the AtCNGC1 channel with the calmodulin-binding domain deleted increased intracellular [K+] of trk1,2. Trk1,2 is hypersensitive to the toxic cations spermine, tetramethylamine, and NH4+. These compounds, as well as amiloride, inhibited trk1,2 growth and thereby improved the efficacy of this yeast mutant as a heterologous expression system for CNGCs. In addition to characterizing mutants of yeast and E. coli as assay systems for plant CNGCs, work presented in this report demonstrates, for the first time, that a plant CNGC can retain ion channel function despite (partial) deletion of its calmodulin-binding domain and that yeast calmodulin can bind to and possibly down-regulate a plant CNGC.

Arabidopsis↗
PubMed1997

Regulated system for heterologous gene expression in Penicillium chrysogenum.

A system for regulated heterologous gene expression in the filamentous fungus Penicillium chrysogenum was established. This is the first heterologous expression system to be developed for this organism. Expression of a recombinant fungal xylanase gene (xylp) and the cDNA for the human tear lipocalin (LCNI) was achieved by placing the encoding sequences under the control of the repressible acid phosphatase gene (phoA) promoter of P. chrysogenum. Secreted recombinant proteins were detected in the growth media of transformed P. chrysogenum cells by means of bioassays, zymogramography, and Western blotting. Levels of transcription and amounts of recombinant proteins secreted varied among transformants, mainly due to the copy number and the integration site of the expression vector on the fungal chromosome.

Carrier Proteins↗
PubMed1999

Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli.

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of Km values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the Km for dopamine under the conditions applied was approximately 1 microM in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with Mr approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations.

Amino Acid Sequence↗
PubMed1996

A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system.

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.

Animals↗
PubMed2004

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors.

BACKGROUND: Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. RESULTS: While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The beta-amino acid taurine possessed 30-50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for beta-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. CONCLUSIONS: Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.

Amygdala↗
PubMed2002

Generating new marine cell lines and transgenic species--conference summary.

Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment. Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others. Much still remains to be learned from these animals, and there is a growing need for new marine models. Models for expression of marine animal genes have been limited to heterologous expression systems. While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible. This has become particularly important with the development of functional genomics in marine models. These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology.

Animals↗
PubMed2006

Expression of recombinant flavin-containing monooxygenases in a baculovirus/insect cell system.

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodopterafrugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.

Animals↗
PubMed2004

Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium.

The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

Amino Acids↗
PubMed2004

Heterologous expression of the modified coat protein of Cowpea chlorotic mottle bromovirus results in the assembly of protein cages with altered architectures and function.

We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host. We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P. pastoris system assemble into viral protein cages with altered architectures and function. This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages. Heterologous expression in P. pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.

Bromovirus↗
PubMed1999

Kv3.1-Kv3.2 channels underlie a high-voltage-activating component of the delayed rectifier K+ current in projecting neurons from the globus pallidus.

The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3. 1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than -10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1-Kv3.2 voltage-gated K+ channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.

Animals↗
PubMed2005

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system.

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.

Animals↗
PubMed1999

A new system for expressing heterologous gene in Escherichia coli regulated by oxygen consistence in the environment.

The expression of Vitreosilla hemoglobin gene (vgb) is regulated by oxygen consistence in E. coli. The gene transcription is activated under the condition of limited oxygen. A new system for expressing heterologous gene in E. coli regulated by dissolved oxygen consistence was constructed. It includes a host bacteria GJ100, which contained T7 RNA polymerase gene controlled by vgb promoter, and an expression vector on which the heterologous gene was under the control of a T7 promoter. The results indicated that E. coli thioredoxin A, IgG binding domain of Staphylococcus protein A(ZZ), snake neurotoxin, salmon calcitonin hexa-polymer, human interleukinII (IL2) and human pro-urokinase genes could be expressed efficiently. Expression level was more than 30% of the total cellular protein.

Bacterial Proteins↗