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Results for “Heterologous expression system”

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At least 163 records · Page 9Linked to original sources
PubMed1997

RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels.

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Acetylcholine↗
PubMed2006

Endoplasmic reticulum-retention C-terminal sequence enhances production of an 11S seed globulin from Amaranthus hypochondriacus in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express an 11S seed globulin from Amaranthus hypochondriacus. Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum-retention signal KDEL, were able to express the proamarantin in P. pastoris. After 48 h of induction, KDEL-proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum-retention C-terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein in P. pastoris. The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.

Allergens↗
PubMed2004

Glucocorticoid-inducible gene expression vectors for use in Drosophila melanogaster Schneider 2 cells.

Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.

Animals↗
PubMed2004

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans.

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) alpha and interleukin (IL) 10 by fusing the coding sequences of mature hTNFalpha and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the DeltatatB and DeltatatC single mutants, indicating that the transport of hTNFalpha and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFalpha and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the DeltatatB mutant than in the wild-type strain.

Escherichia coli Proteins↗
PubMed1993

Identification of a two-component regulatory system controlling methanol dehydrogenase synthesis in Paracoccus denitrificans.

Upstream of the moxFJGIR genes of Paracoccus denitrificans a regulatory region involved in methanol oxidation was identified. The nucleotide sequence of this region was determined and revealed three genes, moxZ, moxY and moxX, which are transcribed opposite to moxF and which encode proteins of 16.4, 48.2 and 24.5 kDa, respectively. Computer alignment analysis revealed that the gene products of moxY and moxX have homology with the protein histidine kinases and the response regulators, respectively, forming the two-component regulatory systems. No significant homology of the moxZ gene product with any known protein, sequenced thus far, was found. The MoxZ, MoxY and MoxX proteins were identified in Escherichia coli in a heterologous expression system. Mutants with an insertion of a kanamycin-resistance marker in moxZ, moxY and moxX were isolated. These mutant strains were unable to grow on methanol while growth on methylamine was not affected. In the moxZ mutant both subunits of methanol dehydrogenase and cytochrome c551i were not synthesized, methanol dehydrogenase activity was absent, and hardly any expression of a moxZ-lacZ transcriptional fusion was found. Complementation of the mutation was observed after addition of the three genes moxZ, Y and X, in trans. This indicates that the two-component regulatory system is involved in activation of the moxF promoter. A mutant with an unmarked deletion in moxZ was isolated. This mutant showed reduced growth on methanol relative to the wild type. Expression of the moxF-lacZ transcriptional fusion gene and methanol dehydrogenase activity in this strain were also lower than those found in the wild type. Therefore, besides the two proteins of the two-component regulatory pair, a third protein, MoxZ, appears to be involved in regulation of methanol dehydrogenase synthesis.

Alcohol Oxidoreductases↗
PubMed2004

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines.

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.

Amino Acid Sequence↗
PubMed2005

Development of PLEX, a plasmid-based expression system for production of heterologous gene products by the gram-positive bacteria Streptococcus gordonii.

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.

Antigens, Bacterial↗
PubMed1992

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.

Animals↗
PubMed2002

Functional improvement of antibody fragments using a novel phage coat protein III fusion system.

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

Base Sequence↗
PubMed2000

Signal transduction by GABA(B) receptor heterodimers.

GABA(B) receptors are G-protein-coupled receptors that mediate inhibition throughout the central and peripheral nervous systems. A single cloned receptor, GABA(B)R1, which has at least three alternatively spliced forms, appears to account for the vast majority of binding sites in the brain for high-affinity antagonists. In heterologous expression systems GABA(B)R1 is poorly expressed on the plasma membrane and largely fails to couple to ion channels. A second gene, GABA(B)R2, which exhibits moderately low homology to GABA(B)R1, permits surface expression of GABA(B)R1 and the appearance of baclofen-sensitive K(+) and Ca(+1) currents. We review the data that supports a model of the native GABA(B) receptor as a heterodimer composed of GABA(B)R1 and GABA(B)R2 proteins. New data from mutagenesis experiments are presented that point to amino acid residues on GABA(B)R1 critical for ligand activation of the heterodimer. The possible role of GABA(B)R2 in signal transduction is also discussed. The interdependent nature of the two subunits for receptor function makes the GABA(B) receptor a useful model to explore the larger significance of GPCR dimerization for G-protein activation.

Amino Acid Sequence↗
PubMed2002

Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum.

Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

Basidiomycota↗
PubMed1997

Development of molecular genetics for Neospora caninum: A complementary system to Toxoplasma gondii.

The development of molecular genetics has greatly enhanced the study of Toxoplasma gondii, and investigations into the biology and pathology associated with neosporosis will be similarly benefited by the development of molecular tools for Neospora caninum. We have demonstrated the feasibility of using the existing DNA vectors developed for T. gondii to transfect and transform the Nc-1 strain of Neospora. We have also shown that T. gondii proteins are faithfully expressed and targeted in N. caninum, indicating the suitability of using Neospora as a heterologous expression system for studying T. gondii. These studies provide the basis for initiating molecular genetic studies on N. caninum and will allow for a number of molecular comparisons of these two closely related, though phenotypically distinct, parasites. Here we describe the methods and reagents used to perform genetic manipulations of N. caninum, and we present some of the principles and potential utilities of these molecular studies, including the use of N. caninum as a heterologous system for the study of T. gondii proteins.

Animals↗
PubMed2002

Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel).

Neuronal members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family of cation channels include the mammalian brain Na(+) channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related alphaENaC or betaENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.

Acid Sensing Ion Channels↗
PubMed1984

Hepatitis B virus transcription in the infected liver.

Hepatitis B virus (HBV) transcription was studied in the liver of an infected chimpanzee and compared with HBV transcription in heterologous systems. Besides the well characterized 2.3-kb surface antigen mRNA produced in most systems, a second major transcript was identified in the liver. This 3.8-kb transcript (+/- 300 bases) is slightly larger than the HBV genome and is probably involved both in core/e antigen synthesis and in HBV replication via reverse transcription. In addition, minor variants of the 2.3-kb surface antigen mRNA were characterized as probably being involved in the expression of HBsAg-related minor proteins. Finally, several potential transcription signals, identified on the HBV genome using heterologous expression systems, were found to be poorly active if at all in the infected liver, thereby stressing the importance of HBV transcription studies performed with liver material.

Animals↗
PubMed1997

Use of a modified bacteriophage to probe the interactions between peptides and ion channel receptors in mammalian cells.

Besides natural peptide ligands, screening of random peptide libraries has yielded novel bioactive peptides for cell surface receptors. A method is described that uses a modified bacteriophage as a detection reagent to monitor the expression of receptor channels in mammalian cells and to probe the molecular interaction between phage-tethered peptides (phi T-peptides) and specific receptor targets. By taking advantage of a specific multivalent interaction between phi T-peptides and the receptor target, assays have been developed that use phi T-peptides specific for the N-methyl-D-aspartate glutamate receptor, an important ligand-gated ion channel in the nervous system, to monitor the receptor expression in cultured mammalian cells. Combining these phi T-peptide binding assays with fluorescence-activated cell sorting, 10(4) random glutamate receptor mutants were screened and candidate interaction residues were identified. This dual heterologous expression system offers a powerful approach to the molecular studies of protein-protein interactions.

Amino Acid Sequence↗
PubMed2001

Molecular characterization and heterologous expression of hypCD, the first two [NiFe] hydrogenase accessory genes of Thermococcus litoralis.

The hypCD genes, encoding the counterparts of mesophilic proteins involved in the maturation of [NiFe] hydrogenases, were isolated from the hyperthermophilic archaeon Thermococcus litoralis. The deduced gene products showed 30-40% identity to the corresponding mesophilic proteins. HypC and HypD were synthesized by the T7 expression system. Heterologous complementation experiments were done in Escherichia coli and Ralstonia eutropha strains lacking functionally active hypC and hypD genes. Only the cytoplasmic hydrogenase of R. eutropha could be processed by HypD from T. litoralis. This was the first demonstration of mesophilic hydrogenase processing using a hyperthermophilic archaeal accessory protein to produce an active enzyme.

Archaeal Proteins↗
PubMed2001

Genetic engineering of phytochrome biosynthesis in bacteria.

The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.

Apoproteins↗
PubMed1998

Cloning and expression of a novel K+ channel regulatory protein, KChAP.

Voltage-gated K+ (Kv) channels are important in the physiology of both excitable and nonexcitable cells. The diversity in Kv currents is reflected in multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes. Given the fundamental importance and diversity of Kv channels, surprisingly little is known regarding the cellular mechanisms regulating their synthesis, assembly, and metabolism. To begin to dissect these processes, we have used the yeast two-hybrid system to identify cytoplasmic regulatory molecules that interact with Kv channel proteins. Here we report the cloning of a novel gene encoding a Kv channel binding protein (KChAP, for K+ channel-associated protein), which modulates the expression of Kv2 channels in heterologous expression system assays. KChAP interacts with the N termini of Kvalpha2 subunits, as well as the N termini of Kvalpha1 and the C termini of Kvbeta subunits. Kv2.1 and KChAP were coimmunoprecipitated from in vitro translation reactions supporting a direct interaction between the two proteins. The amplitudes of Kv2. 1 and Kv2.2 currents are enhanced dramatically in Xenopus oocytes coexpressing KChAP, but channel kinetics and gating are unaffected. Although KChAP binds to Kv1.5, it has no effect on Kv1.5 currents. We suggest that KChAP may act as a novel type of chaperone protein to facilitate the cell surface expression of Kv2 channels.

Amino Acid Sequence↗