Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Heterologous expression system”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed2000

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2.

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.

Adenoviridae↗
PubMedDate not supplied

The use of the rat isolated vagus nerve for functional measurements of the effect of drugs in vitro.

In this article we describe how to dissect, set up and use the rat isolated vagus nerve in a 'grease gap' apparatus which provides a simple and practical method for measuring the effects of drugs on the membrane potential of axons in the nerve in vitro. Some discussion of the origins and development of the technique as well as the strengths and disadvantages of the preparation as a neuropharmacological tool are included. The vagus nerve conducts action potentials in at least three distinct types of axons that can be measured extracellularly as compound action potentials and distinguished on the basis of their conduction velocity and excitability. Activity in myelinated A fibres and unmyelinated C fibres can be measured separately easily. The axons express receptors for a wide range of putative neurotransmitter agents including 5-HT, GABA and ATP as well as other agents such as capsaicin, anandamide, bradykinin and prostanoids. Responses to all of these chemicals can be measured as a depolarization of the nerve fibres. The vagus nerve is an important target for a wide range of drugs and the isolated preparation provides a fairly simple preparation for studying their effects. The isolated vagus nerve is also a convenient system in which the effects of drugs that have been discovered using heterologous expression systems can be assayed on receptors and ion channels that are expressed in a native neural system.

Action Potentials↗
PubMed1998

Expression and characterization of lens membrane intrinsic protein, MIP, in a baculovirus expression system.

PURPOSE: Major intrinsic protein (MIP) is the transmembrane protein in the lens involved in homeostasis of the fiber cell. Although MIP has some intrinsic water and glycerol permeability and can form a gap junction like channels in the reconstituted systems, its role in the lens remained enigmatic. This study is aimed at developing a heterologous expression system for understanding the role of MIP. METHODS: The coding sequence of bovine MIP was subcloned into pBlueBacIII baculovirus transfer vector to facilitate transfer to AcMNPV genome, and the recombinant baculovirus was used to transfect Sf9 cells. Expression of MIP was confirmed by immunoblotting and purified by HPLC. Reconstituted liposomes were used to study the function of the recombinant protein. RESULTS: SDS-PAGE and immunoblots confirmed that the expressed protein was targeted to the cell membrane. MIP accounted for about 10% of total membrane proteins of the transfected Sf9 cell membranes. Molecular size of the recombinant protein is estimated to be about 30-32 kDa on SDS-PAGE, in contrast to 26 kDa of native MIP. The reconstitution studies showed that the channels formed by recombinant MIP are functionally similar to those of native MIP isolated from calf lens membranes. CONCLUSIONS: This study describes functional expression of MIP in a baculovirus expression system. This opens the way to studying the role of MIP and to understanding the effect of mutations of critical residues in the function of MIP.

Animals↗
PubMed2006

HSV-1 based amplicon vectors as an alternative system for the expression of functional HCV proteins.

The lack of efficient systems for the propagation of the hepatitis C virus in vitro, in the past decade, led to the development of several heterologous expression systems for the study of the HCV proteins and the HCV life cycle. HSV-1 amplicon vectors encoding the HCV structural and some of the non structural proteins were generated initially for the expression of high levels of these proteins into mammalian cells. The recent developments in the production of amplicon vectors, allowing the elimination of the contaminating helper HSV-1 virus have given a novel impulse in the study of these vectors as possible vaccine candidates. In this review, an extensive list of the existing amplicon vectors expressing HCV proteins is provided, together with a brief overview of the results obtained by these studies.

Cloning, Molecular↗
PubMed1995

Functional domains in the oxytocin gene for regulation of expression and biosynthesis of gene products.

In the oxytocin (OT) gene several regions can be discerned that have a function in regulating its expression. Firstly, in the proximal 5' flanking region regulatory elements have been discovered that are targets for transcription factors of the nuclear hormone receptor family. Through these elements the OT gene of rat and man is responsive to estrogens, thyroid hormones and retinoids. Furthermore, these elements can be employed by the nuclear hormone orphan receptor family for repressive or inductive actions. In the distal 5' flanking region the POU class III proteins Brn-1, Brn-2, Brn-4, that are expressed in magnocellular neurons, and Oct-6 are able to bind, but do not display a significant regulatory activity on the OT gene in heterologous expression systems. Secondly, the OT precursor harbours both the biologically active hormone and the protein neurophysin that is able to associate with the hormone. Heterologous expression of wild-type and mutant vasopressin cDNAs in peptidergic cell lines shows that the highly homologous vasopressin-associated neurophysin domain associates with the hormone domain within the prohormone. This intramolecular interaction between two prohormone domains serves an essential intracellular function, i.e. the proper sorting of the prohormone into the regulated secretory pathway.

Animals↗
PubMed2003

G protein-coupled receptor overexpression with the baculovirus-insect cell system: a tool for structural and functional studies.

G protein-coupled receptors, whose topology shows seven transmembrane domains, form the largest known family of receptors involved in higher organism signal transduction. These receptors are generally of low natural abundance and overexpression is usually a prerequisite to their structural or functional characterisation. The baculovirus-insect cell system constitutes a versatile tool for the maximal production of receptors. This heterologous expression system also provides interesting alternatives for receptor functional studies in a well-controlled cellular context.

Animals↗
PubMed2003

Electrophysiological analysis of neuronal chemokine receptors.

Several studies have shown that neurons in the central and peripheral nervous systems express a variety of chemokine receptors (CKRs). Activation of these receptors can influence neuronal signaling by regulating synaptic transmission and neuronal excitability. This article presents electrophysiological methods that are currently used to study the normal and pathophysiological role for CKRs in the nervous system. Conventional electrophysiological methods such as patch-clamp recording of isolated neurons, brain slices, and heterologous expression systems are described. In addition, single-cell reverse transcription-polymerase chain reaction is discussed as a technique that can be used in conjunction with patch-clamp recording to further investigate the molecular basis of neuronal CKR activation.

Animals↗
PubMed2004

Structural biology of hepatitis C virus.

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.

Hepacivirus↗
PubMed1990

Antibodies against the large subunit of the EBV-encoded ribonucleotide reductase in patients with nasopharyngeal carcinoma.

The open reading frame corresponding to BORF2 and encoding the large subunit of the Epstein-Barr virus (EBV) ribonucleotide reductase has been inserted into the prokaryotic expression vector pUC19. A 90-kDa protein was produced when the intact plasmid was used as a template for in vitro DNA-directed protein synthesis. Using templates generated by restriction digests within the BORF2 open reading frame, in the same system, truncated polypeptides resulted confirming the identity of the 90-kDa protein. The protein was then produced in a heterologous expression system and used in protein immunoblotting to screen for antibodies in sera from nasopharyngeal carcinoma (NPC), Burkitt's lymphoma (BL) or control subjects. Twenty out of 33 NPC sera were positive for antibodies against the large subunit, 13 of these were positive for both IgG and IgA, whilst 7 were positive for IgG only. Out of 15 BL sera and 10 control sera, none were positive. These results are similar to those observed for other EBV-encoded enzymes, including the DNase which had been used as an early marker for the development of NPC. The results presented here indicate that antibodies against the large subunit of ribonucleotide reductase could serve as an additional marker for NPC.

Antibodies, Neoplasm↗
PubMed1996

Baculovirus-based expression of mammalian caveolin in Sf21 insect cells. A model system for the biochemical and morphological study of caveolae biogenesis.

Caveolae were originally defined morphologically as 50-100 nm noncoated vesicular organelles located at or near the plasma membrane. Caveolin, a vesicular integral membrane protein of 21 kDa, is a principal protein component of caveolae membranes in vivo. Caveolin interacts with itself to form high molecular mass oligomers, suggesting that it might play a structural role in the formation of caveolae membranes. However, it remains controversial whether recombinant expression of caveolin is necessary or sufficient to generate caveolae membranes in vivo. To directly address this issue, we have taken a different experimental approach by exploiting a heterologous expression system. Here, we have recombinantly expressed mammalian caveolin in Sf21 insect cells using baculovirus-based vectors. Two isoforms of caveolin have been identified that differ at their extreme N terminus; alpha-caveolin contains residues 1-178, and beta-caveolin contains residues 32-178. After recombinant expression in Sf21 insect cells, both alpha- and beta-caveolin formed SDS-resistant high molecular mass oligomers of the same size as native caveolin. Morphologically, expression of either caveolin isoform resulted in the intracellular accumulation of a homogeneous population of caveolae-sized vesicles with a diameter between 50 and 120 nm (80.3 +/- 14.8 nm). This indicates that each caveolin isoform can independently generate these structures and that caveolin residues 1-31 are not required for this process. Using caveolin as a marker protein and a detergent-free procedure to purify caveolae from mammalian cells, we purified these recombinant caveolin-induced vesicles from insect cells. These purified recombinant vesicles: (i) have the same buoyant density as mammalian caveolae; (ii) appear as approximately 50-100 nm membranous structures by whole-mount electron microscopy; and (iii) contain approximately 95% of the recombinantly expressed caveolin protein by Western blotting. Immuno-labeling of these structures with anti-caveolin IgG confirmed that they contain caveolin. Thus, ectopic overexpression of caveolin in this heterologous system is sufficient to drive the formation of caveolae-like vesicles. Further functional analysis demonstrated that caveolin was capable of interacting with a known caveolin-interacting protein, Ha-Ras, when coexpressed in insect cells by co-infection with two recombinant baculoviruses. Taken together, our results demonstrate that baculovirus-based expression of caveolin in insect cells provides an attractive experimental system for studying the biogenesis of caveolae.

Animals↗
PubMed1998

Connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access.

We reported previously (Falk, M. M., Kumar, N. M., and Gilula, N. B. (1994) J. Cell Biol. 127, 343-355) that the membrane integration of polytopic connexin polypeptides can be accompanied by an inappropriate cleavage that generates amino-terminal truncated connexins. While this cleavage was not detected in vivo, translation in standard cell-free translation/translocation systems resulted in the complete cleavage of all newly integrated connexins. Partial cleavage occurred in heterologous expression systems that correlated with the expression level. Here we report that the transmembrane topology of connexins generated in microsomal membranes was identical to the topology of functional connexins in plasma membranes. Characterization of the cleavage site and reaction showed that the connexins were processed by signal peptidase immediately downstream of their first transmembrane domain in a reaction similar to the removal of signal peptides from pre-proteins. Increasing the length and hydrophobic character of the signal anchor sequence of connexins completely prevented the aberrant cleavage. This result indicates that their signal anchor sequence was falsely recognized and positioned as a cleavable signal peptide within the endoplasmic reticulum translocon, and that this mispositioning enabled signal peptidase to access the cleavage sites. The results provide direct evidence for the involvement of unknown cellular factors in the membrane integration process of connexins.

Amino Acid Sequence↗
PubMed1997

Alpha3, beta2, and beta4 form heterotrimeric neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different beta subunits assemble with an alpha subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for alpha3, beta2, and beta4 subunits. Beta2 and beta4, in pairwise combination with alpha3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). Alpha3beta4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through alpha3beta2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for alpha3, beta2, and beta4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both beta2 and beta4 subunits in functional receptors.

Alkaloids↗
PubMed2000

Molecular cloning of a Na+-dependent nucleoside transporter from rabbit intestine.

PURPOSE: Substantial species differences in the transport kinetics of nucleosides and therapeutic nucleoside analogs have been observed in various experimental systems. To explain these differences at a molecular level, it is necessary to clone the relevant transporters and examine their functional characteristics in heterologous expression systems. The goal of the present study was to clone the nucleoside transporters present in rabbit, an important preclinical animal model, and to functionally characterize the clone(s). METHODS: A Polymerase Chain Reaction (PCR)-based homology cloning approach in conjunction with Rapid Amplification of cDNA Ends (RACE) was used to isolate a full-length cDNA. Characterization of this transporter was accomplished through heterologous expression in Xenopus laevis oocytes. RESULTS: A full-length cDNA encoding a purine-selective, Na+-dependent nucleoside transporter, rbSPNT1, was isolated from rabbit small intestine. The encoded protein is 658 amino acid residues in length. Hydropathy analysis suggests that rbSPNT1 has 11 to 14 transmembrane domains. In Xenopus laevis oocytes expressing rbSPNT1, the uptake of uridine and inosine was enhanced significantly; uridine transport was inhibited by purine, but not pyrimidine nucleosides. mRNA transcripts for rbSPNT1 were detected primarily in intestine, lung, and kidney and at lower levels in liver, brain, and heart. CONCLUSIONS: A full-length functional nucleoside transporter was cloned. Sequence analysis and functional characterization suggest that rbSPNT1 is the rabbit homolog of the purine-selective nucleoside transporter, N1. The cloned rbSPNT1 can be used to understand the molecular mechanisms responsible for the observed species differences in the transport of nucleosides and therapeutic nucleoside analogs.

Amino Acid Sequence↗
PubMed2004

Coupling of human nicotinic acetylcholine receptors alpha 7 to calcium channels in GH3 cells.

The neuronal nicotinic acetylcholine receptor alpha7 (nAChR alpha7) may be involved in cognitive deficits in Schizophrenia and Alzheimer's disease. A fast pharmacological characterization of homomeric alpha7 receptors is mostly hampered by their low functional expression levels in heterologous expression systems. In the present study expression of homomeric nAChR alpha7 was achieved in GH3 rat pituitary cells. Alpha7 subunits were heterologously expressed as components of [125I]-labeled alpha-bungarotoxin binding nAChRs (Bmax: 1.2 pmol/mg protein). Function of the expressed alpha7 ion channels was assessed by patch-clamp recording and calcium imaging. While acetylcholine-induced currents desensitized within much less than 1 s, calcium-sensitive fluorescence transients peaked after 5-10 s and returned to background levels within 30 s only. The fluorescence signal was blocked by isradipine and removal of extracellular sodium indicated that in these cells opening of rapidly desensitizing alpha7 nAChR triggers calcium influx via voltage-gated, DHP-sensitive calcium channels. In this cellular system, agonists revealed the following rank order of potency: epibatidine>anatoxin A>AAR17779>ABT-594>DMPP>nicotine>GTS-21>cytisine>ABT-418>acetylcholine>choline>ABT-089. All of the signals were inhibited by the alpha7 antagonists alpha-bungarotoxin (pIC50: 7.4) and methyllycaconitine (pIC50: 7.8). Further, marketed antidepressants showed antagonistic activity with the following rank order of potency: fluoxetine>imipramine>paroxetine>sertraline. These data illustrate that coupling to voltage-gated calcium channels allows a rapid and reliable functional examination of nAChR alpha7.

Animals↗
PubMed2004

Determinants of the sensitivity of AMPA receptors to xenon.

BACKGROUND: There is substantial and growing literature on the actions of general anesthetics on a variety of neurotransmitter-gated ion channels, with the greatest attention being focused on inhibitory gamma-amino butyric acid type A receptors. In contrast, glutamate receptors, the most important class of fast excitatory neurotransmitter-gated receptor channels, have received much less attention, and their role in the production of the anesthetic state remains controversial. METHODS: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors formed from a variety of different subunits were expressed in Xenopus oocytes and HEK-293 cells, and their sensitivities to the inhalational general anesthetics xenon, isoflurane, and halothane were determined using two-electrode voltage clamp and patch clamp techniques. The effects of desensitization on anesthetic sensitivity were investigated using cyclothiazide and site-directed mutagenesis. An ultrarapid application system was also used to mimic rapid high-concentration glutamate release at synapses. RESULTS: The authors show that xenon can potently inhibit AMPA receptors when assayed using bath application of kainate. However, when the natural neurotransmitter l-glutamate is used under conditions in which the receptor desensitization is blocked and the peak of the glutamate-activated response can be accurately measured, the pattern of inhibition changes markedly. When desensitization is abolished by a single-point mutation (L497Y in GluR1 and the equivalent mutation L505Y in GluR4), the xenon inhibition is eliminated. When AMPA receptors are activated by glutamate using an ultrarapid application system that mimics synaptic conditions, sensitivity to xenon, halothane, and isoflurane is negligible. CONCLUSIONS: AMPA receptors, when assayed in heterologous expression systems, showed a sensitivity to inhalational anesthetics that was minimal when glutamate was applied rapidly at high concentrations. Because these are the conditions that are most relevant to synaptic transmission, the authors conclude that AMPA receptors are unlikely to play a major role in the production of the anesthetic state by inhalational agents.

Anesthetics, Inhalation↗
PubMed2005

Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression.

The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation, was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type 2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late endosomal and lysosomal compartments after treatment of the cells with compounds that correct the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH, while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this heterologous expression system recapitulates the defective processing and mistrafficking of tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells. Coexpression of other melanosomal proteins in this heterologous system may further aid our understanding of the details of normal and pathologic processing of melanosomal proteins.

Albinism, Oculocutaneous↗
PubMed1995

Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides.

One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies. In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb. sphaeroides mutants; DBC omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting. Thus, we have created a heterologous expression system which supports the assembly of a functional "foreign" light-harvesting complex. This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system.

Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide.

Amino Acid Sequence↗