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PubMed1995

Kinetic, circular dichroism and fluorescence studies on heterologously expressed carnitine palmitoyltransferase II.

Km estimates for carnitine and palmitoyl-CoA of heterologously expressed rat liver carnitine palmitoyl-transferase-II (rCPT-II) were 950 +/- 27 microM and 34 +/- 6 microM, respectively. Vmax for the enzyme was 1.8 mumol/min/mg purified protein. Consistent with an ordered reaction mechanism in which palmitoyl-CoA binds first, SDZ CPI 975, a reversible carnitine palmitoyltransferase inhibitor containing both carnitine and alkyl moieties, inhibited rCPT-II competitively with carnitine and uncompetitively with palmitoyl-CoA. Substrate-enzyme interactions were examined by circular dichroism (CD) and fluorescence. Both carnitine and palmitoyl-CoA alone induced conformational changes in the enzyme; dissociation constant estimates by CD for carnitine and palmitoyl-CoA were 41 +/- 5 microM and 7 +/- 2 microM, respectively.

Animals↗
PubMed1996

Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells.

The single channel properties of four P2X receptors heterologously expressed in Chinese hamster ovary cells were determined from outside-out patch recordings. P2X1 and P2X4 channels opened in brief flickery bursts with conductances of approximately 18 and 9 pS respectively. P2X2 receptors had a single channel conductance of approximately 21 pS. Openings of P2X3 channels were too rapid to be reliably resolved. Application of ATP to macro-patches expressing multiple P2X2 or P2X4 receptors evoked sustained currents, in contrast responses of P2X1 or P2X3 receptors desensitised during continued agonist application. These data show that four different homomeric P2X receptors form ion channels with distinct single channel properties.

Adenosine Triphosphate↗
PubMed2000

A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum.

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha-(2-aminoethoxyvinyl) glycine (AVG).

Alphaproteobacteria↗
PubMed1994

Activation of heterologously expressed D3 dopamine receptors: comparison with D2 dopamine receptors.

Recombinant rat D3 dopamine receptors heterologously expressed in Chinese hamster ovary (CHO) cells are functionally coupled to endogenous G proteins. The affinity of the receptors for agonists is regulated by guanine nucleotides in the same manner as that of other G protein-linked receptors. The magnitude of the change in affinity induced by GTP is much less, however, than what is observed for recombinant rat D2 receptors expressed in CHO cells at similar densities. The striking difference is that the low affinity state (uncoupled D3 receptors) has a much higher affinity for agonists than does the low affinity state (uncoupled) of D2 receptors. Both receptors in the high affinity state (G protein coupled) have similar affinities for dopamine. Three functional responses result from activation of D3 or D2 receptors expressed in CHO cells. Both receptor subtypes mediate inhibition of adenylyl cyclase activity, increases in extracellular acidification rates that are prevented by removal of external Na+ and by amiloride analogs, and stimulation of cell division. However, these three functional results of D3 and D2 receptor activation are both quantitatively and qualitatively different. Dopamine activation of D3 receptors is always 2-5-fold less efficacious than dopamine activation of D2 receptors, despite similar densities of receptors. Both D3 and D2 receptor-mediated increases in extracellular acidification rates are blocked by pertussis toxin; however, the D3 response and not the D2 response is partially attenuated by membrane-soluble cAMP analogs. D3 and D2 receptor-mediated stimulation of mitogenesis is blocked by pertussis toxin and unaffected by cAMP analogs. The results show that D2 and D3 dopamine receptors mediate similar signaling events and are additional examples of G protein-linked receptors that can activate more than one pathway. Having functionally coupled D2 and D3 receptors expressed in the same cell type enabled determinations of agonist potencies at both D2 and D3 receptors. Comparison of the potencies at the two receptors reveals that none of the agonists is as selective for D3 receptors as was previously thought based on radioligand binding data.

Animals↗
PubMed2000

Heterologous expression of Trypanosoma cruzi trans-sialidase in Leishmania major enhances virulence.

Earlier studies showed that mice primed for a few hours with the trans-sialidase (TS) of Trypanosoma cruzi, the agent of Chagas' disease, become highly susceptible to trypanosomal infection. These studies suggest that TS affects parasite virulence independent of antigenic stimulation. Potentially, TS could enhance or reduce the virulence of heterologous microbes depending on the mechanism of TS action and on the type of immune response elicited by the particular parasite. We tested this hypothesis by expressing heterologous TS in Leishmania major, a protozoan parasite that causes cutaneous leishmaniasis and lacks TS and the TS product alpha2-3-linked sialic acid. Leishmania cells transfected with a T. cruzi TS expression construct made high levels of active enzyme, which was present in the promastigotes and shed into the extracellular milieu. TS expression did not affect L. major binding to and entry into cultured macrophages or its tropism for macrophage infection in vivo. However, TS-expressing L. major exhibited elevated virulence in BALB/c mice, as determined by lesion progression, parasite numbers, and macro- and microscopic examination of cutaneous lesions. Several genetic tests proved that the enhanced virulence was directly attributable to TS expression. The results are consistent with TS functioning to sabotage the mouse immune system to confer a growth advantage on T. cruzi and transgenic L. major. These data suggest that heterologous expression of T. cruzi virulence factors in Leishmania may provide a new approach for dissecting their function in vivo.

Animals↗
PubMed2006

Heterologous expression of lipoprotein-associated phospholipase A2 in different expression systems.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a key enzyme involved in atherosclerosis, and has been considered as a new target for drug discovery. The major difficulty for high-throughput screening of Lp-PLA(2) inhibitors and for functional studies was their fast and efficient production. Purification of native Lp-PLA(2) from human plasma was complicated and produced a very low yield. We herein examined the feasibility of expressing and purifying recombinant Lp-PLA(2) in different heterologous expression systems. The fusion Lp-PLA(2) was expressed at high levels and exhibited strong enzyme activity in insect cell-baculovirus expression system. The functional enzyme could also be produced in Pichia pastoris. The inclusion of a Kozak sequence increased greatly the expression level of recombinant Lp-PLA(2) in insect cells, but had little effect on the expression of recombinant Lp-PLA(2) in P. pastoris and Escherichia coli. P. pastoris-produced Lp-PLA(2) could be purified rapidly and conveniently through a one-step procedure, while baculovirus-produced Lp-PLA(2) could be efficiently purified through a two-step procedure. This ability to readily produce recombinant Lp-PLA(2) could provide a screening model for Lp-PLA(2) inhibitors and will facilitate further studies on this enzyme.

Animals↗
PubMed2001

Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells.

The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca(2+), Mn(2+) and Na(+), have a high selectivity for Na(+) compared with Ca(2+), and are inhibited by Gd(3+) (half-maximal inhibition at 1 microM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca(2+) concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)(+) RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca(2+) inflow and a modest enhancement of thapsigargin-initiated Ca(2+) inflow (measured using fura-2) and no enhancement of the highly Ca(2+)-selective store-operated Ca(2+) current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na(+) and Ca(2+), and are inhibited by Gd(3+) (half-maximal inhibition at 3 microM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na(+) compared with Ca(2+); (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca(2+)-selective store-operated Ca(2+) channels.

Animals↗
PubMed1997

Heterologous expression and biochemical characterization of two functionally different type I fatty acid synthases from Brevibacterium ammoniagenes.

The coryneform bacterium, Brevibacterium ammoniagenes, contains two structurally related but functionally differentiated type I fatty acid synthases, FAS-A and FAS-B. Isolation of homogeneous preparations of both enzymes was achieved by constructing specific fasA and fasB expression systems. In B. ammoniagenes, insertional mutagenesis of fasB allowed the specific production of enzymatically active FAS-A. The corresponding fasA mutant was not suited for FAS-B purification as the level of this enzyme was extremely low, in the fasA-disruptants. Instead, FAS-B could be efficiently expressed in the heterologous host, Escherichia coli. Using specific antisera against each of the two FAS variants, FAS-A was shown to be the predominant FAS protein in B. ammoniagenes. In contrast the two enzymes are expressed at comparable rates in E. coli even though the same upstream sequences were associated with fasA and fasB, as in B. ammoniagenes. Due to their differential capacities of being activated to the phosphopantetheine-containing holo-enzyme in the heterologous host, only FAS-B but not FAS-A exhibited overall FAS activity when isolated from E. coli. Irrespective of their origin, the purified FAS-A and FAS-B proteins were indistinguishable with respect to their flavin fluorescence, their subunit size and their sucrose density gradient sedimentation characteristics. Nevertheless, the in vitro products of both enzymes differ characteristically: while FAS-A synthesizes mainly the 18-carbon fatty acids oleate and stearate with only traces of palmitate, the major product of FAS-B is palmitic acid. No unsaturated fatty acids are produced by FAS-B. Thus, the two B ammoniogenes type I fatty acid synthases differ, in spite of their very similar overall protein structure, in both their ability to synthesize oleic acid and in their chain-length specificities.

Brevibacterium↗
PubMed2002

Codon optimization improves heterologous expression of a Schistosoma mansoni cDNA in HEK293 cells.

Differences in codon usage can seriously hamper the expression of cloned cDNAs in heterologous systems. In this study, we show that the expression of a cloned Schistosoma mansoni cDNA in cultured HEK293 cells was dramatically increased by rewriting a portion of the cDNA according to human preferred codon usage, suggesting that codon optimization is a valuable strategy for improving the heterologous expression of helminth sequences. We further describe a simple modification of a recursive PCR-based method, which allows the rewriting of long stretches of DNA sequence in a single PCR reaction. This method can be used to optimize the codon usage of virtually any DNA from helminths and other parasites.

Animals↗
PubMed1992

Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin.

Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions. An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.

Azurin↗
PubMed1994

Studies on the importance of microsomal epoxide hydrolase in the detoxification of arene oxides using the heterologous expression of the enzyme in mammalian cells.

In order to investigate the role of the microsomal epoxide hydrolase (mEH) in the detoxification of arene oxides in the presence of a high endogenous glutathione S-transferase (GST) activity-a situation found in several organs--we expressed the rat mEH cDNA in BHK21 Syrian hamster cells. These cells have high GST activities but contain an extremely low endogenous mEH enzyme activity. We obtained several cell clones which expressed the mEH heterologously, as determined by immunoblotting. The cell clone BHK21-mEH/Mz1 had the highest level of mEH protein. Immunofluorescence showed that the level of expression was almost homogeneous throughout the cell population. Total protein isolated from the cell line BHK21-mEH/Mz1 had a specific mEH activity of 123 pmol/min/mg protein, as determined with benzo[a]pyrene 4,5-oxide (B[a]P 4,5-oxide), which was 60 times higher than the activity in the parental cell line and eight times lower than the activity found in rat hepatocytes. However, BHK21-mEH/Mz1 cell homogenates were found to catalyze the conjugation of B[a]P 4,5-oxide to glutathione extremely well. The ratio of the GST enzyme activity to the mEH enzyme activity towards this substrate was 23 in the BHK21-mEH/Mz1 cell line. For hepatocytes this ratio was only six. Despite their already high potential to inactivate B[a]P 4,5-oxide by conjugation to glutathione, BHK21-mEH/Mz1 cells were better protected against the toxic and mutagenic effects of B[a]P 4,5-oxide than the parental cell line due to the expression of the mEH. The mEH, however, failed to protect the cells from the toxic and mutagenic effects of the bay region epoxide anti-7-methylbenz[a]anthracene-3,4-diol 1,2-oxide.

Animals↗
PubMed1994

Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture.

The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvolfia serpentina and the berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes.

Amino Acid Sequence↗
PubMed2005

Modulating effect of ginseng saponins on heterologously expressed HERG currents in Xenopus oocytes.

AIM: To examine the effects of ginseng saponins on the heterologously expressed human ether-a-go-go related gene (HERG) that encodes the rapid component of the delayed rectifier K+ channel. METHODS: A two-electrode voltage clamp technique was used. HERG currents were recorded in Xenopus oocytes injected with HERG cRNA. RESULTS: Crude saponins of Korean red ginseng (GS) induced a minimal increase of the maximal HERG conductance without changes in the voltage-dependent HERG current activation and inactivation curves. GS, however, decelerated HERG current deactivation in a concentration-dependent manner, which was more noticeable with panaxitriol (PT) than panaxidiol (PD). Consistently, ginseng saponins increased the HERG deactivation time constants with the order of potency of Rg1 (a major component of PT)>Rf1>Rb1 (a major component of PD). Re had little effect on HERG deactivation. During a cardiac action potential, GS increased the outward HERG current. CONCLUSION: Ginseng saponins enhance HERG currents, which could be in part a possible mechanism of the shortening cardiac action potential of ginseng saponins.

Action Potentials↗
PubMed2001

Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans.

BACKGROUND: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis. RESULTS: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose. CONCLUSIONS: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

Anthelmintics↗
PubMed1999

Heterologous expression and product identification of Colletotrichum lagenarium polyketide synthase encoded by the PKS1 gene involved in melanin biosynthesis.

The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible alpha-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN.

Aspergillus oryzae↗
PubMed2006

Marker and promoter effects on heterologous expression in Aspergillus nidulans.

To study the effects of selection marker, promoter type, and copy number on heterologous expression in Aspergillus nidulans, strains were constructed with single- and multicopy plasmid integrations bearing a reporter gene (lacZ) under the control of either an inducible (alcA) or constitutive (gpdA) promoter and one of three Aspergillus nutritional marker genes (argB, trpC, or niaD). beta-Galactosidase activity in the transformants varied over three orders of magnitude, with the majority of levels in the range of 5x10(3)-1x10(4) U/mg. Significant differences in mean expression levels were found when comparing single-copy transformants with the same promoter but a different marker. Transformants with the argB marker had the highest average expression, approximately threefold over the trpC or niaD clones. For each promoter, maximal expression for the set was seen in the range of the single-copy clones, implying that increasing the copy number does not reliably increase expression in Aspergillus.

Aspergillus nidulans↗
PubMed2003

Odorant response assays for a heterologously expressed olfactory receptor.

Odorant responsiveness of a mouse olfactory receptor, mOR-EG, was investigated in various heterologous cells using a variety of detection methods. Odorant-induced Ca(2+) response was observed in HEK293 cells that coexpressed mOR-EG and the promiscuous G protein, G alpha 15. Without G alpha 15, a robust increase in cAMP level was observed upon odorant-stimulation in various mammalian cells. A luciferase reporter gene assay using zif268 promoter was adopted to amplify the cAMP signals. In Xenopus laevis oocytes, odorant-stimulated currents were recorded when mOR-EG cRNA was co-injected with either G alpha 15 or cAMP-dependent channel. These results suggest that odorant responsiveness can be monitored via a signaling pathway mediated by endogenous G alphas or transfected G alpha 15 in heterologous cell systems. Various functional assays for a heterologously expressed olfactory receptor reported in this study, are potentially useful for high-throughput ligand screening and functional analyses of hundreds of olfactory receptors.

Animals↗
PubMed2006

Electrophysiological effect of fluoxetine on Xenopus oocytes heterologously expressing human serotonin transporter.

AIM: To investigate the electrophysiological effect of fluoxetine on serotonin transporter. METHODS: A heterologous expression system was used to introduce human serotonin transporter (hSERT) into Xenopus oocytes. A 2-electrode voltage clamp technique was used to study the pharmacological properties of fluoxetine. RESULTS: hSERT-expressing oocytes were perfused with 10 micromol/L serotonin (5-HT) to induce hSERT-current. The 5-HT-induced hSERT currents were dose-dependently reversed by fluoxetine. The RC50 (concentration that achieved a 50% reversal) was approximately 3.12 micromol/L. Fluoxetine took more time to combine with hSERT than 5-HT did, and it was also slow to dissociate from hSERT. This long-lasting effect of fluoxetine affected normal 5-HT transport. Fluoxetine significantly prolonged the time constant for 5-HT-induced hSERT current. These results might be used to explain the long-lasting anti-anxiety effect of fluoxetine in clinical practice, because it increases the concentration of 5-HT in the synaptic cleft by its enduring suppression of the function of 5-HT transporters. CONCLUSION: Fluoxetine inhibits 5-HT reuptake by competing with 5-HT and changing the normal dynamics of hSERT.

Animals↗