Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding human N-myristoyltransferase (hNMT) was cloned into the overexpression vector pT7-7 which utilizes the T7 RNA polymerase gene expression system. The hNMT enzyme was purified to near homogeneity with more than 95% recovery using a single-step purification method involving SP-Sepharose fast flow column chromatography. The specific activity of the purified NMT was 220 nmol/min/mg of protein in the presence of oncoprotein-derived peptide substrate pp60src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS-polyacrylamide gel electrophoresis. Antibodies to Escherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The over-expressed hNMT was homogeneous and showed enzyme activity.
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PMID:Overexpression of human N-myristoyltransferase utilizing a T7 polymerase gene expression system. 877 63

The elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was expressed in Escherichia coli and purified. The SsEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase. Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from the E. coli S-100 extract by a two-step procedure. From 1 litre of cell culture, about 2 mg of purified recSsEF-1 alpha was obtained. The N-terminal sequence of the first 30 amino acid residues of recSsEF-1 alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1 alpha was Ser instead of Met. The M(r) of recSsEF-1 alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1 alpha (SsEF-1 alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were identical. Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity [Masullo, De Vendittis and Bocchini (1994) J. Biol. Chem. 269, 20376-20379] with the same efficiency as that displayed by SsEF-1 alpha.
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PMID:Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus. 886 95

The goal of the next generation of cancer chemotherapy is effective tumor-selectivity. A tumor-selective target with high therapeutic potential is the elevated methionine requirement of tumor cells relative to normal cells. We have termed the elevated requirement for methionine in tumors methionine dependence. To selectively target the methionine dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine alpha-deamino-gamma-mercaptomethane-lyase (methioninase, METase) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). The METase gene was then ligated into the pT7-7 overexpression plasmid containing the T7 RNA polymerase promoter and recloned in E. coli strain BL21(DE3). The pAC-1 clone was isolated by its yellow-orange color which is due to high enrichment of the pyridoxal phosphate-containing recombinant methioninase (rMETase) and distinguished rMETase-overproducer from rMETase-negative colonies. A scale-up production protocol which contained a heat step, two DEAE Sepharose FF ion-exchange, and one ActiClean Etox endotoxin-affinity chromatography columns has been established. The pAC-1 clone produces rMETase at approximately 10% of the total soluble protein and up to 1 g/liter in shake-flask culture. The protocol can produce therapeutic rMETase at the multi-gram level per batch with high yield (> 60%), high purity (> 98%), high stability, and low endotoxin. Purified rMETase is stable to lyophilization. The t1/2 of rMETase was 2 h when rMETase was administered by i.v. injection in mice. Studies of the antitumor efficacy of rMETase in vitro and in vivo on human tumors xenografted in nude mice demonstrated that all types of human tumors tested including those from lung, colon, kidney, brain, prostate, and melanoma were sensitive to rMETase. In contrast, normal cells were insensitive to rMETase in vitro and correspondingly, no toxicity was detected in vivo at the effective doses. In conclusion, the overexpression clone and large-scale production protocols for rMETase have enabled rMETase to be used as a tumor-selective therapeutic with broad indication and high promise for effective, low-toxicity human cancer therapy.
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PMID:Overexpression and large-scale production of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane-lyase for novel anticancer therapy. 905 89

Synthetic genes offer many potential advantages over conventional plasmid DNA, such as simplicity in purification, absence of endotoxin contamination, and more importantly, flexibility in chemical modifications to render them specific properties. We have used PCR amplified fragments as a model to test the feasibility of using synthetic genes for gene therapy. The CAT reporter gene driven by the CMV promoter (CMV-CAT), ie a nuclear expression system, or by the bacteriophage T7 promoter (T7-CAT), ie a cytoplasmic expression system, was used to evaluate this concept. The expression efficiency of both plasmids (pUCCMV-CAT and pT7-CAT) and their corresponding PCR fragments (fCMV-CAT and fT7-CAT) were compared on a molar basis. Limited expression of CAT was found with fCMV-CAT. However, fT7-CAT consistently gave a CAT activity comparable to that of pT7-CAT. When fT7-CAT was codelivered with pCMV/T7-T7pol (a self-amplifying-T7 RNA polymerase autogene), high CAT activity could be detected up to 9 days. This expression was much longer than the duration of expression with a nuclear expression system. These encouraging results imply that gene therapy with synthetic genes could be both feasible and efficient.
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PMID:Delivery of a PCR amplified DNA fragment into cells: a model for using synthetic genes for gene therapy. 927 22

Escherichia coli strain PEF42 produces a sodium-ion-dependent hybrid F1F0-ATPase consisting of the Propionigenium modestum subunits a, b, c and delta, of a hybrid alpha subunit and of the E. coli subunits beta, gamma and epsilon. The gene encoding subunit c of the P. modestum F1F0-ATPase was cloned into the pT7-7 expression vector to yield plasmid pT7c. E. coli PEF42 was transformed with plasmid pT7c together with plasmid pGP1-2, which harbours the gene for the T7 RNA polymerase. The production of the P. modestum subunit c was induced by a temperature shift from 30 degrees C to 42 degrees C for 30 min and led to an increased concentration of this protein in the membrane of the host strain. The c subunit produced in E. coli moved as a monomer in dodecylsulfate electrophoresis. The protein was extracted from the cells with chloroform/methanol, purified and incorporated into sodium dodecylsulfate micelles. Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature-stable spectrum (between 20-60 degrees C) with a high proportion of alpah-helical structure. Upon incubation of subunit c with [14C]dicyclohexylcarbodiimide the protein became labelled in a sodium-ion-dependent manner, similar to the labelling observed if the purified F1F0-ATPase of P. modestum, was treated with the radioactive carbodiimide. The Na+-specific site was therefore retained in the isolated c subunit dissolved in dodecylsulfate.
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PMID:Subunit c from the sodium-ion-translocating F1F0-ATPase of Propionigenium modestum--production, purification and properties of the protein in dodecylsulfate solution. 928 3

This paper reports a novel expression system constructed to maximize the production in Escherichia coli of d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We produced a recombinant plasmid by the insertion of the cDNA encoding for the RgDAAO into the multiple cloning site of the expression vector pT7.7 (pT7-DAAO), downstream of the T7 RNA polymerase binding site. The pT7-DAAO, which encodes a fully active fusion protein with six additional residues at the N-terminus of DAAO, was used to transform the BL21(DE3) and BL21(DE3)pLysS E. coli cells. In the latter host and under optimal IPTG induction conditions, soluble and active chimeric DAAO was expressed in these cells up to 930 U/g of cell (and a fermentation yield of 2300 U/liter of fermentation broth), with a specific activity of 8.8 U/mg protein. RgDAAO represents approximately 8% of the total soluble protein content of the cell.
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PMID:Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis d-amino acid oxidase. 979 Aug 93

Inefficient nuclear transport of plasmid DNA continues to be a problem in nonviral vector-mediated gene transfer. This has made the cytoplasmic expression system an increasingly attractive idea. We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter. The pCMV/T7-T7pol autogene does not encounter the problems associated with previously used autogenes. For instance, pCMV/T7-T7pol is easily amplified and purified from bacteria. Furthermore, the CMV promoter is used to drive the first round of synthesis of T7 RNA polymerase, thus negating the use of purified enzyme in the transfection complex. The endogenous T7 RNA polymerase produced from the CMV promoter could then act on the T7 promoter of pCMV/T7-T7pol in an autoregulatory mechanism. pCMV/T7-T7pol induces higher, more sustained levels (> 7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C- or with the nuclear expression system pCMV-CAT. This seems to be due to the high levels of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol. This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter as compared with the nuclear CMV promoter in pCMV/T7-T7pol. Therefore, pCMV/T7-T7pol could replace existing autogenes for regeneration of T7 RNA polymerase and efficient target gene expression.
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PMID:A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes. 1043 11

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.
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PMID:Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs. 1043 89

There are two types of infection caused by pathogenic microorganisms, intracellular infection and intercellular infection. Infection of pathogenic leptospira is an intercellular infection. The immunological reaction of host to intercellular infection is unique. The potential immunogen of an expressed protein should meet three criteria: it can be degraded (by antigen-present cells in the host); it should have antigenic epitope which can be recognized by specific antibodies and have at least one epitope that can be recognized by an MHC II protein and T cell receptor. In this study we report the cloning of an L. interrogans protein in plasmid rpDJt and the immunogencity of the expressed protein derivative. A genomic library of L. interrogans serovar lai strain 017 was constructed with the plasmid vector pUC18. Recombinant plasmids, designated pDJH2 and pDJ8 were screened from the bank. EcoRI-inserted fragment of 1. 9 kb recombinant DNA of pDJH2 was ligated into T7 RNA polymerase/promoter vectors (pT7-7). Then they were transformed into E. coli JM109 (De3), one of subclones, designated rpDJt was achieved. SDS-PAGE showed that the molecular weights of expression proteins were 68 kd and 23 kd respectively, designated p68 and p23. Purifying and isolating p68 and p23, we separated them from SDS-Polyacrylamide gels by using Side-Strip method. After fragmenting and electroeluting, p68 and p23 were injected into guinea pigs and rabbits. An extremely strong immune response to p68 was obtained since an anti-p68 antibody response could be detected to a dilution 1:524,288 (guinea pigs) and 1:262,144 (rabbits) by ELISA while anti-P23 antibody being 1:1024 (the same to guinea pigs and rabbits). The results of improved MTT and conA 3HTdR transformation methods showed the activities and proliferation of Th-cells were increased in guinea pigs after p68 immunization (IL-6, 83.25 IU/ml, IL-2, 28.75 IU/ml; RPI, 2.04, SI, 65.62%) Thlymphocyte existed in two subclasses, the Th1- and Th2-cells. A major role of Th2-cells is to "help" B-cells differentiate, replicate, and secrete antibody. The properties of these interactions explain why p68 makes good antigen and p23 does not. The antigens responsible for eliciting the production of protective antibodies are not known; however, several outer membrane proteins on L. interrogans are candidates for vaccine. Our results suggest that expresion protein p68 from recombinants (rpDJt) may be a candidate for gene engineered subunit vaccine for Leptospirosis.
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PMID:[Immunogenecity of expressed protein p68 from recombinant plasmid rpDJt in L. interrogans serovar lai]. 1068 17

A gene (pueA, polyurethane esterase A) encoding an extracellular polyurethanase (PueA) was cloned from Pseudomonas chlororaphis into Escherichia coli. The enzyme secreted from E. coli showed esterase activity when assayed with p-nitrophenyl acetate. Subcloning of a 3. 2-kb SalI-EcoRI fragment into a T7 RNA polymerase expression vector (pT7-6) produced a (35)S-labeled protein of 65 kDa. Nucleotide sequencing of pueA showed an open reading frame encoding a 65-kDa protein of 617 amino acid residues, with the serine hydrolase consensus sequence GXSXG. PueA was over-expressed using the pT7-6 vector transformed into E. coli BL21(DE3) and was purified in one step using Sephadex G-75.
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PMID:The polyester polyurethanase gene (pueA) from Pseudomonas chlororaphis encodes a lipase. 1075 42


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