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Results for “Heterologous expression”

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PubMed2005

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin.

BACKGROUND: Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. RESULTS: In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. CONCLUSION: The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Animals↗
PubMed1996

Heterologous expression of full-length and truncated forms of the recombinant guluronate-specific alginate lyase of Klebsiella pneumoniae.

The alyA gene, which encodes a guluronate-specific alginate lyase from Klebsiella pneumoniae, was cloned into the plasmid pCT54 to facilitate heterologous expression of the enzyme in Escherichia coli. The greatest enzyme specific activity was observed when the complete gene and flanking regions of DNA (contained within a 1.95 kb HindIII fragment) were cloned in the correct orientation relative to the vector-encoded trp promoter. Heterologous expression of the intact gene complete with flanking regions resulted in a 20-fold increase in enzyme yield compared to the original construct, pRC5. PCR mutagenesis and/or restriction endonuclease digestion was used to generate a series of fragments containing either the whole or truncated versions of the gene. Active enzyme was produced from constructs in which the region encoding the signal peptide had been deleted, although the recombinant protein was retained within the bacterial cells. An internal methionine (position 74) could be used as a start site for translation but resulted in the accumulation of inactive protein within inclusion bodies.

Amino Acid Sequence↗
PubMed2005

Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Delta). The expression of the DhGPD1 gene in the gpd1Delta background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

Gene Expression Regulation, Fungal↗
PubMed1996

Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum.

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion.

Amino Acid Sequence↗
PubMed2002

Heterologous expression of dihydroflavonol 4-reductases from various plants.

Dihydroflavonol 4-reductases (DFR) catalyze the stereospecific reduction of dihydroflavonols to the respective flavan 3,4-diols (leucoanthocyanidins) and might also be involved in the reduction of flavanones to flavan-4-ols, which are important intermediates in the 3-deoxyflavonoid pathway. Several cDNA clones encoding DFR have been isolated from different plant species. Despite the important function of these enzymes in the flavonoid pathway, attempts at heterologous expression of cDNA clones in Escherichia coli have failed so far. Here, three well known heterologous expression systems for plant-derived genes were tested to obtain the functional protein of DFR from Gerbera hybrids. Successful synthesis of an active DFR enzyme was achieved in eukaryotic cells, using either baker's yeast (Saccharomyces cerevisiae) or tobacco protoplasts (Nicotiana tabacum), transformed with expression vectors containing the open reading frame of Gerbera DFR. These expression systems provide useful and powerful tools for rapid biochemical characterization, in particular the substrate specificity, of the increasing number of cloned DFR sequences. Furthermore, this tool allows the stereospecific synthesis of (14)C-labeled leucoanthocyanidins in high quality and quantity, which is a prerequisite for detailed biochemical investigation of the less understood enzymatic reactions located downstream of DFR in anthocyanin, catechin and proanthocyanidin biosynthesis.

Alcohol Oxidoreductases↗
PubMed2004

Heterologous expression of barley and wheat oxalate oxidase in an E. coli trxB gor double mutant.

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.

Amino Acid Sequence↗
PubMed2006

Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance.

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.

Amino Acid Sequence↗
PubMed2000

Nutrient transporter function studied in heterologous expression systems.

Although a large number of plasma cell nutrient transport proteins has been cloned in the last couple of years, much remains to be learned about their structure-function relationships, membrane topology, posttranslational regulation, and bioenergetics of transport. Major progress in the study of the human and animal transporters has come from heterologous expression systems, which offer the benefits of ease of genetic selection and manipulation, short generation time of the organisms in which transporters are expressed, and comparatively high levels of expression of the recombinant proteins. Because our main focus is mammalian peptide transporters, the intestinal peptide transporter, PEPT1, and its renal counterpart, PEPT2, will serve here as models for the analysis of their structure and function when they are heterologously expressed in different cell systems.

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↗
PubMed1994

The heterologous expression and characterization of human prostaglandin G/H synthase-2 (COX-2).

The open reading frame of human cyclooxygenase-2 was cloned by pcr amplification of IL-1 beta stimulated human dermal fibroblast cDNA. The coding region was used to construct a recombinant baculovirus which when used to infect Sf9 cells directed the expression of recombinant human cyclooxygenase-2. The heterologously expressed enzyme was characterized and found to display all salient features of cyclooxygenase. Large-scale microsomal preparations of infected cells yielded more than 20 units of enzyme with a specific activity of 240 nmoles prostaglandin product/mg protein.

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↗
PubMed2003

Substrate specificity and kinetic properties of seven heterologously expressed dog cytochromes p450.

Seven dog cytochromes p450 (p450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 microM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 microM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 microM), similar to that in dog liver microsomes (Km = 2.3 microM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 microM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16alpha-, 2alpha/2beta-, and 6beta-positions. Selectivity of CYP3A12 was observed toward testosterone 6beta-(Km = 83 microM) and 2alpha/2beta-hydroxylations (Km = 154 microM). However, the 16alpha-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 microM for CYP2C21). Therefore, the 6beta- and 16alpha-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the p450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog p450s and the development of dog p450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.

Animals↗
PubMed2006

Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118.

Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

Anti-Bacterial Agents↗
PubMed2007

Functional analysis of Kluyveromyces lactis carboxylic acids permeases: heterologous expression of KlJEN1 and KlJEN2 genes.

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1delta cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae.

Carboxylic Acids↗
PubMed1995

Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera.

A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)- and (S)-N-methylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.

Alcohol Oxidoreductases↗
PubMed1999

Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors.

The suramin analogue 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X1-P2X4 as well as heteromultimeric P2X2/P2X3 receptors has been characterized. P2X1 receptors were most sensitive to inhibition by NF023 with IC50 values of 0.24 and 0.21 microM for the rat and human homologue, respectively. P2X3 receptors have an intermediate sensitivity with IC50 values of 8.5 and 28.9 microM for rat and human subtypes, respectively and P2X2 was the least sensitive subtype (IC50 > 50 microM). P2X4 receptors were insensitive to NF023 at concentrations up to 100 microM. Coexpression of rat P2X3 with rat P2X2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X3 receptors (alphabeta meATP as agonist; IC50 = 1.4 and 1.6 microM, respectively). NF023 inhibited P2X1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 microM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP (K(B) = 1.1 +/- 0.2 microM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X1 receptors heterologously expressed in Xenopus oocytes.

Adenosine Triphosphate↗
PubMed2003

Specific lipid requirements of membrane proteins--a putative bottleneck in heterologous expression.

Membrane proteins are mostly protein-lipid complexes. For more than 30 examples of membrane proteins from prokaryotes, yeast, plant and mammals, the importance of phospholipids and sterols for optimal activity is documented. All crystallized membrane protein complexes show defined lipid-protein contacts. In addition, lipid requirements may also be transitory and necessary only for correct folding and intercellular transport. With respect to specific lipid requirements of membrane proteins, the phospholipid and glycolipid as well as the sterol content of the host cell chosen for heterologous expression should be carefully considered. The lipid composition of bacteria, archaea, yeasts, insects,Xenopus oocytes, and typical plant and mammalian cells are given in this review. A few examples of heterologous expression of membrane proteins, where problems of specific lipid requirements have been noticed or should be thought of, have been chosen.

Animals↗
PubMed1992

cDNA sequence and heterologous expression of the human neurokinin-3 receptor.

Functional cDNA clones encoding the human neurokinin-3 receptor were isolated from human brain mRNA. The cloned human neurokinin-3 receptor was expressed in COS cells and Xenopus oocytes, where peptide binding affinity and intracellular effector activation were determined. Neurokinin B is the most potent agonist, followed by eledoisin, substance K and substance P. The binding affinities of these peptides at the human neurokinin-3 receptor differ quantitatively from the rat receptor, implying a functional consequence of the sequence divergence between the two species. Heterologous expression in oocytes revealed that, unlike the neurokinin-1 receptor, the efficacy of ion channel activation mediated by the neurokinin-3 receptor does not approximate the binding affinity. The heterologous expression of the human neurokinin-3 receptor will facilitate further investigation into its biochemical functions.

Amino Acid Sequence↗