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PubMed1998

D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme.

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

Base Sequence↗
PubMed1994

Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene.

Heterologous expression of the Clostridium cellulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTL500E).

Biodegradation, Environmental↗
PubMed2004

Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23.

The voltage-gated sodium channel NaV1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/NaV1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant NaV1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat NaV1.8 (rNaV1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat NaV1.8 Na+ currents were recorded (789 +/- 89 pA, n=62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNaV1.8 (mean IC50 value 12.5 microM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNaV1.8 (r=0.72, P<0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta1 and beta3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNaV1.8 is discussed. We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNaV1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNaV1.8 as novel analgesics.

Animals↗
PubMed1997

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.

Alanine Dehydrogenase↗
PubMed1999

Optimized heterologous expression of glutathione reductase from Cyanobacterium anabaena PCC 7120 and characterization of the recombinant protein.

Glutathione reductase (GR) from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli SG5. Silent random mutations were introduced in the 5' region of DNA encoding the enzyme in order to generate a high-level expression clone. To maximize protein expression, the culture conditions were also optimized. In the high-level expression clones selected, E. coli-preferred codons were selectively used at certain positions. Under the optimal expression conditions, a yield of 17 mg recombinant protein per liter was obtained, which is about 10-fold higher than that of the wild-type enzyme. A hexahistidine tag was added at the C-terminal of the protein in order to allow IMAC affinity purification. This strategy simplified the purification process and provided a homogeneous enzyme for functional characterization. Anabaena GR uses NADPH as a coenzyme, like most of the GRs from other sources, but the KM values for NADPH and GSSG are higher than those of enzymes previously studied. The Anabaena enzyme also shows significant activity when NADH is used as a reductant.

Amino Acid Sequence↗
PubMed1999

Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells.

1. We combined patch clamp and fura-2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2. ATP, bradykinin and intracellular InsP3 activated a non-selective cation current (IhTRP3) in htrp3-transfected CPAE cells but not in non-transfected wild-type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3-transfected cells than in control CPAE cells. 3. The permeability for monovalent cations was PNa > PCs approximately PK >> PNMDG and the ratio PCa/PNa was 1.62 +/- 0.27 (n = 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist-activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24.4 +/- 0.7 microM). 4. The single-channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5. Thapsigargin and 2,5-di-tert-butyl-1, 4-benzohydroquinone (BHQ), inhibitors of the organellar Ca2+-ATPase, failed to activate IhTRP3. U-73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor-sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6. It is concluded that hTRP3 forms non-selective plasmalemmal cation channels that function as a pathway for agonist-induced Ca2+ influx.

Adenosine Triphosphate↗
PubMed1993

Cloning, heterologous expression, and chromosomal localization of human inositol polyphosphate 1-phosphatase.

Inositol polyphosphate 1-phosphatase, an enzyme in the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1 position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. We used a cDNA that encodes bovine inositol polyphosphate 1-phosphatase as a probe to isolate the human counterpart by low-stringency hybridization. The 1.74-kb human cDNA has 341 bp of 5' untranslated region, 180 bp of 3' untranslated region, poly(A)32, and predicts a protein of 399 amino acids. Human and bovine inositol polyphosphate 1-phosphatases show 84% amino acid sequence identity. Northern blot analysis from a variety of human tissues demonstrates that a 1.9-kb mRNA is ubiquitously expressed with highest levels in pancreas and kidney. Several higher molecular weight mRNAs also are expressed in brain, muscle, heart, and liver. We have confirmed the functional identity of the human cDNA by heterologous expression in NIH 3T3 fibroblasts, COS-7 cells and Escherichia coli. Polymerase chain reaction assay of a panel of human-rodent somatic cell hybrid DNA using human inositol polyphosphate 1-phosphatase-specific DNA primers resulted in amplification of a specific product using chromosome 2 DNA as template. Fluorescence in situ hybridization of metaphase chromosomes localizes the gene to chromosome 2 band q32. The identification of the human inositol polyphosphate 1-phosphatase gene locus provides a target for linkage analysis to identify defects in patients with inherited psychiatric disorders that respond to lithium ions, an inhibitor of the enzyme.

3T3 Cells↗
PubMed1998

Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3.

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

Copper↗
PubMed1998

Cloning and heterologous expression of genes from the kinamycin biosynthetic pathway of Streptomyces murayamaensis.

The genes for most of the biosynthesis of the kinamycin antibiotics have been cloned and heterologously expressed. Genomic DNA of Streptomyces murayamaensis was partially digested with MboI and a library of approximately 40 kb fragments in E. coli XL1-BlueMR was prepared using the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes. After transferal of these clusters to S. lividans ZX7, expression of one cluster was established by HPLC with photodiode array detection. Peaks were identified from the kin cluster for dehydrorabelomycin, kinobscurinone, and stealthin C, which are known intermediates in kinamycin biosynthesis. Two shunt metabolites, kinafluorenone and seongomycin were also identified. The structure of the latter was determined from a quantity obtained from large-scale fermentation of one of the clones.

Anti-Bacterial Agents↗
PubMed2006

Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus.

Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the myxothiazol biosynthetic genes in M. xanthus results in the production of myxothiazol in yields comparable to the natural producer strain.

Bacterial Proteins↗
PubMed1998

Heterologous expression in Escherichia coli of soluble active-site random mutants of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 by coexpression of molecular chaperonins GroEL/ES.

A system for heterologous expression in Escherichia coli of dehaloalkane dehalogenase Dh1A from Xanthobacter autotrophicus strain GJ10 is presented. The strategy involved overexpression of E. coli chaperonins GroEL/ES which facilitated the production of soluble Dh1A. When active-site mutant forms were constructed they could not to any detectable degree be expressed in a soluble state in the absence of overproduced GroEL/ES. However, with the described expression system, wild-type Dh1A as well as variant forms randomly mutated in the active-site residues Phe172 and Trp175 were reliably produced. An introduced C-terminal (His)5-tag provided an immunological handle as well as a site for metal ion coordination utilized in affinity chromatography for the purification of recombinant Dh1A. The purified His-tagged enzyme, Dh1A-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.

Binding Sites↗
PubMed1996

Heterologous expression and characterisation of mouse brain fatty acid binding protein.

A novel brain-type member of the fatty acid binding protein family (B-FABP) was heterologously expressed in Escherichia coli, either as inclusion bodies at 37 degrees C or in soluble form at 22 degrees C. Both B-FABP renatured from inclusion bodies and the solubly expressed protein could be purified to homogeneity by anion exchange chromatography and gel filtration in a functional conformation as they bound oleic acid with high affinity. None of the five cysteines of B-FABP was involved in disulphide bond formation. Isoelectric focusing revealed heterogeneity of the renatured protein but not of the solubly expressed protein. By Western blotting using affinity purified rabbit antibodies raised against the recombinant B-FABP it was demonstrated that in adult mice, B-FABP is predominantly expressed in the olfactory bulb.

Amino Acid Sequence↗
PubMed1996

Optimized heterologous expression of the polymorphic human glutathione transferase M1-1 based on silent mutations in the corresponding cDNA.

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.

Amino Acid Sequence↗
PubMed2005

Physiological regulation, xenobiotic induction, and heterologous expression of P450 monooxygenase gene pc-3 (CYP63A3), a new member of the CYP63 gene cluster in the white-rot fungus Phanerochaete chrysosporium.

In order to characterize the functional diversity in CYP63 cluster of tandemly linked P450 genes (pc-1, pc-2, and pc-3) in Phanerochaete chrysosporium, here we report the functional characterization of pc-3 (CYP63A3), a newly cloned member of this group. pc-3 expression was favored in nutrient-limited versus nutrient-rich media in 3-6-day-old cultures and was upregulated by starch as a carbon source or by oxygenation of cultures. pc-3 was induced by various xenobiotics in defined nutrient-limited (3-9-fold) and nutrient-rich (2-5-fold) cultures. Particularly, a range of unsubstituted and substituted aliphatic hydrocarbons (alkanes and fatty acids) induced the expression under the two nutrient conditions albeit in a differential manner. Interestingly, pc-3 was also inducible by certain oxygenated mono aromatics (nitrophenol, benzoate, and resorcinol), lower molecular weight (2 to 4 ring size) polycyclic aromatic hydrocarbons (PAHs) and alkali-treated lignin derivatives in nutrient-rich malt extract cultures. The study further establishes that the three CYP63 genes (CYP63A1, A2, and A3) are independently regulated despite being members of the tandem gene cluster with high gene structural similarity (13-14 introns) and protein sequence homology (59-85%). The pc-3 cDNA (1,812 bp) was expressed in E. coli as a His-tagged protein (approximately 74 kDa). This constitutes the first report on heterologous expression of a P450 monooxygenase enzyme from this model white-rot fungus.

Adaptation, Physiological↗
PubMed2004

[Cloning and heterologous expression of a novel delta6 -desaturase gene from Rhizopus arrhizus NK030037].

A 593 bp DNA fragment was amplified from Rhizopus arrhizus NK030037 with degenerate oligonucleotide primers designed based on the sequences information for fungi delta6-fatty acid desaturase genes by RT-PCR and sequenced. Gene specific primers derived from this partial sequence were used for the amplification of the 3'- and 5'-ends of this cDNA by RACE method, and this lead to a full-length cDNA sequence of 1 482 bp was amplified. Sequence analysis showed this cDNA sequence had an open reading frame(ORF) of 1 377 bp coding 458 amino acids of 52 kD. The deduced amino-acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. The full-length cDNA sequence is a putative novel delta6-fatty acid desaturase gene. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA RAD6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L. Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters(FAME) were analyzed by gas chromatography(GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 3.85%. Gas chromatography-mass spectrometry( GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the transgenic product exhibited delta6-fatty acid desaurase activity, converting LA to GLA specifically.

Amino Acid Sequence↗
PubMed2003

Heterologous expression of a ketohexokinase in potato plants leads to inhibited rates of photosynthesis, severe growth retardation and abnormal leaf development.

In the present paper we investigated the effect of heterologous expression of a rat liver ketohexokinase in potato (Solanum tuberosum L.) plants with the aim of investigating the role of fructose 1-phosphate in plant metabolism. Plants were generated that contained appreciable activity of ketohexokinase but did not accumulate fructose 1-phosphate. They were, however, characterised by a severe growth retardation and abnormal leaf development. Studies of (14)CO(2) assimilation and metabolism, and of the levels of photosynthetic pigments, revealed that these lines exhibited restricted photosynthesis. Despite this fact, the levels of starch and soluble sugars remained relatively constant. Analysis of intermediates of starch and sucrose biosynthesis revealed large increases in the triose phosphate and fructose 1,6-bisphosphate pools but relatively unaltered levels of inorganic phosphate and 3-phosphoglycerate, and these lines were also characterised by an accumulation of glyceraldehyde. The transformants neither displayed consistent changes in the activities of Calvin cycle enzymes nor in enzymes of sucrose synthesis but displayed a metabolic profile partially reminiscent of that brought about by end-product limitation, but most likely caused by an inhibition of photosynthesis brought about by the accumulation of glyceraldehyde. Analysis of the metabolite contents in lamina and vein fractions of the leaf, and of the enzymes of carbohydrate oxidation indicate that the phloem-enriched veins of ketohexokinase-expressing leaves tend toward hypoxia and indicate a problem of phloem transport.

Animals↗
PubMed1999

Heterologous expression of a green fluorescent protein-pertussis toxin S1 subunit fusion construct disrupts calcium channel modulation in rat superior cervical ganglion neurons.

The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1-100 ng/microl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca2+ channel modulation. The heterologously expressed toxin retained specificity for G alpha(i/o)-dependent pathways as VIP-mediated modulation of N-type Ca2+ channels and muscarine-mediated inhibition of M-type K+ channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.

Animals↗
PubMed2004

Modulation of ion channels and synaptic transmission by a human sensory neuron-specific G-protein-coupled receptor, SNSR4/mrgX1, heterologously expressed in cultured rat neurons.

Human sensory neuron-specific G-protein-coupled receptors (SNSRs) are expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages. The neuronal function of human SNSRs is difficult to assess because rodent orthologs have yet to be clearly defined, and individual isoforms are found only in a small subset of primary sensory neurons. To circumvent this problem, we expressed human SNSR4 (hSNSR4; also known as Hs.mrgX1) in rat superior cervical ganglion (SCG), dorsal root ganglion (DRG), and hippocampal neurons using nuclear injection or recombinant adenoviruses and examined modulation of ion channels and neurotransmission using whole-cell patch-clamp techniques. BAM8-22 (a 15 amino acid C-terminal fragment of bovine adrenal medulla peptide 22), a peptide agonist derived from proenkephalin, inhibited high (but not low) voltage-activated Ca2+ current in both DRG and SCG neurons expressing hSNSR4, whereas no response was detected in control neurons. The Ca2+ current inhibition was concentration dependent and partially sensitive to Pertussis toxin (PTX) treatment. Additionally, the peptide was highly effective in modulating current arising from M-type K+ channels in SCG neurons expressing hSNSR4. In hippocampal neurons expressing hSNSR4, BAM8-22 induced presynaptic inhibition of transmission that was abolished after PTX treatment. Our data indicate that hSNSR4, when heterologously expressed in rat neurons, can be activated by an opioid-related peptide, couples to G(q/11)-proteins as well as PTX-sensitive G(i/o)-proteins, and modulates neuronal Ca2+ channels, K+ channels, and synaptic transmission.

Animals↗