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PubMed2004

Palmitoylputrescine, an antibiotic isolated from the heterologous expression of DNA extracted from bromeliad tank water.

Heterologous expression of large fragments of microbial DNA extracted directly from environmental samples (environmental DNA, or eDNA) in easily cultured hosts should provide access to some of the natural products produced by previously uncultured bacteria. The natural product antibiotic palmitoylputrescine (1) was isolated from Escherichia coli transformed with a cosmid (pCSLF16) containing DNA extracted directly from Costa Rican bromeliad tank water. In this report we describe the characterization of this antibiotic and its biosynthetic gene.

Anti-Bacterial Agents↗
PubMed1997

Biotin sulfoxide reductase. Heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 micromol of biotin sulfoxide reduced per min/nmol of enzyme and Km values of 29 and 15 microM for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.

Chromatography, High Pressure Liquid↗
PubMed2005

Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways.

The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery. It enables the (over)production of structurally complex substances through transfer of the biosynthetic genes from the original producer to more amenable heterologous hosts, and provides the basis to generate novel analogs through biosynthetic engineering. Furthermore, the lateral transfer of 'silent' (not expressed under standard laboratory conditions) secondary metabolite pathways or metagenomic DNA into surrogate host strains is expected to yield new, potentially bioactive compounds. This review discusses recent reports on the heterologous production of natural products with emphasis on polyketide and nonribosomally biosynthesized peptide compounds.

Bacteria↗
PubMed2006

Heterologous expression of membrane and soluble proteins derepresses GCN4 mRNA translation in the yeast Saccharomyces cerevisiae.

This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2alpha (eIF-2alpha) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4p is a transcription factor initially found to be required for transcriptional induction of genes responsible for amino acid or purine biosynthesis. Using various GCN4-lacZ fusions, knockout yeast strains, and anti-eIF-2alpha-P/anti-eIF-2alpha antibodies, we observed that heterologous expression of the membrane-bound alpha1beta1 Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase alpha1 subunit derepressed GCN4 mRNA translation up to 70-fold. GCN4 translation was very sensitive to the presence of heterologous protein, as a density of 1 per thousand of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation to the same extent. In situ activity of the heterologously expressed protein was not required for derepression of GCN4 mRNA translation, as illustrated by the expression of an enzymatically inactive Na,K-ATPase. Two- to threefold overexpression of the highly abundant and plasma membrane-located endogenous H-ATPase also induced GCN4 translation. Derepression of GCN4 translation required phosphorylation of eIF-2alpha, the tRNA binding domain of Gcn2p, and the ribosome-associated proteins Gcn1p and Gcn20p. The increase in Gcn4p density in response to heterologous expression did not induce transcription from the HIS4 promoter, a traditional Gcn4p target.

Alcohol Oxidoreductases↗
PubMed2003

Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.

Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled iodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1992

Heterologous expression of an alginate lyase gene in mucoid and non-mucoid strains of Pseudomonas aeruginosa.

A 1.95 kb DNA fragment containing the aly gene from Klebsiella pneumoniae, which encodes an alginate lyase, has been ligated into the broad-host-range vector pLAFR3. Transfer of the resultant recombinant plasmid, pALY8, into mucoid and non-mucoid strains of Pseudomonas aeruginosa resulted in expression of the alginate lyase. The heterologously expressed alginate lyase, which had the same isoelectric point and substrate specificity as the native enzyme, altered the morphology of mucoid strains. Analysis of the extracellular material from mucoid strains revealed that lyase expression reduced the M(r) and overall yield of alginate produced. The mature form of the recombinant enzyme was the same as that produced extracellularly by Klebsiella pneumoniae; however, most of the alginate lyase was retained intracellularly by P. aeruginosa.

Alginates↗
PubMed2003

Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein.

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.

Aminohydrolases↗
PubMed1996

Heterologous expression and characterization of rat liver glucokinase regulatory protein.

Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414-419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Cloning, characterization and heterologous expression of the Blakeslea trispora gene encoding orotidine-5'-monophosphate decarboxylase.

The pyrG gene of the fungus Blakeslea trispora, encoding orotidine-5'-monophosphate decarboxylase (OMPD) enzyme, was cloned by heterologous hybridization of a genomic library with the Mucor circinelloides pyrG gene. The deduced amino acid sequence of the B. trispora pyrG gene is highly similar to the OMPD from other organisms. Hybridization analyses revealed that the only copy of this gene present in the genome of B. trispora is constitutively expressed. Heterologous complementation of a mutant of M. circinelloides deficient in OMPD activity with the B. trispora pyrG gene and promoter sequence confirmed the function of this gene. This functional complementation demonstrates that heterologous expression in M. circinelloides might be used to investigate the function of genes of B. trispora.

Amino Acid Sequence↗
PubMed1998

Heterologous expression and coupling of G protein-gated inwardly rectifying K+ channels in adult rat sympathetic neurons.

1. G protein-gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole-cell patch-clamp technique. 2. Following coinjection of either GIRK1-2 or GIRK1-4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA-activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed. 3. NA-mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin-sensitive pathway suggesting coupling through Galphas. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP-mediated GIRK channel currents when compared with untreated cells. 4. Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine M to PTX-treated neurons) failed to elicit overt GIRK channel currents. 5. GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA-mediated inhibition of Ca2+ channels was significantly attenuated. 6. In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor-G protein-effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.

Animals↗
PubMed2002

Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B subtilis/E coli shuttle vector system.

A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins.

Anti-Bacterial Agents↗
PubMed2003

[Active drug delivery by heterologous expression of membrane transporters].

The oligopeptide transporter PEPT1 is predominantly expressed in the brush-border membranes of small intestinal epithelial cells, where it plays pivotal roles in the efficient absorption of di-/tripeptides. PEPT1 has enormous potential as an oral drug delivery target, because it also mediates the intestinal absorption of peptide-mimetic and nonpeptide substrates. We demonstrated that the peptide derivation of amino acid-related drugs is applicable to improve their intestinal absorption. We have found that oligopeptide transport activity is also expressed in cancer cell lines. The tissue distribution of bestatin was significantly increased in solid tumors that overexpress PEPT1 in nude mice. Orally administered bestatin strongly suppressed tumor growth. These results provided the first demonstration of the tumor-selective delivery of a drug by specific transport activity. The absolute degree of PEPT1 mRNA expression in the small intestine was determined using real-time PCR in rats. Starvation of the animals increased the mRNA expression level profoundly in the upper small intestine. The longitudinal expression pattern was well correlated with the intestinal transport of cefadroxil in rats. We constructed a recombinant adenovirus vector encoding PEPT1 cDNA. Heterologous expression of PEPT1 in the liver was successfully achieved by simple intvavenous. Administration of the vector, resulting in increased liver distribution of [3H] carnosine. In situ perfusion of the brain with the vector doubled the brain distribution of cefadroxil. Heterologous expression of the drug transporter in vivo could be a useful approach for drug delivery.

Adenoviridae↗
PubMedDate not supplied

Vaccines against enterotoxigenic bacterial pathogens based on hybrid Salmonella that express heterologous antigens.

In this report, we examine two aspects in the development of a vaccine against enterotoxigenic bacterial pathogens based on hybrid Salmonella that express heterologous antigens. First, we describe the construction of a non-toxic fusion peptide for immunization against Escherichia coli that produce heat-labile (LT) and heat-stable (ST) enterotoxins. For that construction, the 5' terminus of the gene coding for ST was fused to the 3' terminus of the gene coding for the binding subunit of LT(LT-B). The ST gene was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion. Maximum expression of ST antigenicity was obtained when a seven-amino-acid proline-containing linker was included between the LT-B and ST moieties. The purified LT-B/ST fusion peptide consisted of a single polypeptide chain with an apparent molecular weight of 18,000. The LT-B/ST fusion peptide was non-toxic and immunologic determinants of both LT and ST were recognized by antibodies directed against the native toxins. Animals immunized with either crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST. The second aspect reported here examines a mechanism for stabilizing expression of heterologous antigens in attenuated Salmonella mutants by integration of the heterologous gene (LT-B) into the chromosome of the carrier. A comparative in vitro study of the levels of expression of LT-B between the cointegrate strain and an isogenic strain carrying the LT-B gene on a multicopy plasmid demonstrated that the initial levels of expression of both strains is similar, that the plasmid-carrying strain loses the ability to express the heterologous antigen very quickly and that the cointegrate continues to maintain and express the antigen without the requirement for a stabilizing antibiotic.

Animals↗
PubMed2006

Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads.

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.

Acyltransferases↗
PubMed2005

The Synthetic Gene Designer: a flexible web platform to explore sequence manipulation for heterologous expression.

"Codon optimization" is a general approach to improving heterologous expression where genes are moved from their native genomes into alternatives that exhibit different patterns of codon usage. However, despite reports of successful manipulations and the existence of stand-alone codon optimization software packages or commercial services that offer to redesign genes, the scientific community lacks any systematic understanding of what exactly it means to optimize codon usage. Thus we present a bona fide web application, the "Synthetic Gene Designer," which contrasts with existing software by providing a centralized, free, and transparent platform for the broader scientific community to develop knowledge about synthetic gene design. Consistent with this goal, our software is associated with a moderated e-forum that promotes discussion of synthetic gene design and offers technical support. In addition, the Synthetic Gene Designer presents enhanced functionality over existing software options: for example, it enables users to work with non-standard genetic codes, with user-defined patterns of codon usage and an expanded range of methods for codon optimization. The Synthetic Gene Designer, together with on-line tutorials and the forum, is available at .

Animals↗
PubMed1998

Heterologous expression of genes encoding bacterial light-harvesting complex II in Rhodobacter capsulatus and Rhodovulum sulfidophilum.

In the present work we report the high-level expression of foreign genes encoding the light-harvesting (LHII) membrane-spanning polypeptides in photosynthetic bacteria. To do this we first constructed three deletion strains of Rhodovulum (Rhv.) sulfidophilum in which all or part of the puc operon, encoding the peripheral light-harvesting proteins, is missing. To investigate the heterologous expression of the light-harvesting polypeptides from Rb. capsulatus in Rhv. sulfidophilum and vice versa we have reintroduced functional foreign LH genes into these and equivalent strains of Rhodobacter (Rb.) capsulatus. In some cases very high levels of expression were obtained (85%) of those observed in the wild type), while in other cases much lower expression was observed; possible reasons for these differences are discussed. The heterologously expressed proteins were shown to contain normal pigment-binding sites and to be normally and functionally integrated within the host photosynthetic apparatus. The results indicate that heterologous proteins are able to assemble properly and enter into the same protein-protein interactions as their analogs originally present in the host strain.

Bacterial Proteins↗
PubMed1996

Inter-individual variability in the oxidation of 1,2-dibromoethane: use of heterologously expressed human cytochrome P450 and human liver microsomes.

1,2-Dibromoethane (1,2-DBE) is mainly used as an additive in leaded gasoline and as a soil fumigant and it is a suspected carcinogen in humans. In this study, the oxidative bioactivation of 1,2-DBE to 2-bromoacetaldehyde (2-BA) was studied using heterologously expressed human cytochrome P450 (P450) isoenzymes and human liver microsomes. Out of ten heterologously expressed human P450 isoenzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2C9, CYP2C18, CYP3A4 and CYP3A5), only human CYP2A6, CYP2B6 and CYP2E1 metabolized 1,2-DBE, albeit with strongly differing catalytic efficiencies. The apparent Km and Vmax values were 3.3 mM and 0.17 pmol/min per pmol P450 for CYP2A6, 9.7 mM and 3.18 pmol/min per pmol P450 for CYP2B6 and 42 microM and 1.3 pmol/min per pmol P450 for CYP2E1, respectively. In all of 21 human liver samples studied, 1,2-DBE was oxidized with activities ranging from 22.2 to 1027.6 pmol/min per mg protein, thus showing a 46-fold inter-individual variability. The kinetics of the oxidative metabolism of 1,2-DBE to 2-BA in human liver microsomes were linear, indicating the involvement of primarily one single P450 isoenzyme. There was a tendency towards a positive correlation between the oxidative metabolism of 1,2-DBE in the human liver microsomes and the 6-hydroxylation of chlorzoxazone, a selective substrate for CYP2E1. Furthermore, the oxidative metabolism of 1,2-DBE was inhibited by the specific CYP2E1 inhibitors disulfiram (DS) and diethyldithiocarbamate (DDC). In contrast, a poor correlation was found between the immunochemically quantified amount of CYP2E1 and the microsomal chlorzoxazone 6-hydroxylation or the 1,2-DBE oxidation. The results indicate that CYP2E1 is probably the major P450 isoenzyme involved in the oxidative hepatic metabolism of 1,2-DBE in humans. The inter-individual variability in the oxidative bioactivation of 1,2-DBE in humans, largely due to inter-individual variability in the catalytic activity of hepatic CYP2E1, may have important consequences for the risk assessment for human exposure to 1,2-DBE.

Acetaldehyde↗
PubMed1991

Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus.

The cDNA clone lambda ML-1 encoding one of the extracellular lignin peroxidases from the white rot fungus, Phanerochaete chrysosporium, was heterologously expressed in an active form using a recombinant baculovirus system. The glycosylated extracellular form of the recombinant protein contained the ferriprotoporphyrin IX moiety and was capable of oxidizing both iodide and the model lignin compound, veratryl alcohol. In comparative peroxidase assays using guaiacol and Mn(II), the recombinant lignin peroxidase did not appear to be Mn(II) dependent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the heterologously expressed peroxidase had an apparent molecular weight similar to that of the native fungal isozyme H8. The elution profile of the active recombinant enzyme derived by ion-exchange chromatography and immunoblot analysis using an anti-H8 monoclonal antibody provided further evidence that the lambda ML-1 DNA encodes the lignin peroxidase H8.

Baculoviridae↗