Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.
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PMID:Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency. 839 44

Supercoil-stabilized intramolecular triplexes have been described under various conditions in different polypurine.polypyrimidine sequences such as (dG)n.(dC)n and mixed sequences including d(GA)n.d(CT)n while information about the triplexes in (dA)n.(dT)n is scarce. Using osmium tetroxide complexes and diethyl pyrocarbonate as structural probes, we show a pyrimidine.purine.pyrimidine (TAT) triplex in (dA)58.(dT)58 sequence in a supercoiled plasmid pE19. Strong modification of approximately six central thymines and approximately six T's at the 3'-end of the (dT)58 stretch as well as the DEPC modification of the 5'-half of the (dA)58 strand suggested the prevalence of the H-y3 triplex conformer. At native superhelix density, optimum conditions for the triplex formation were close to 1 mM MgCl2, pH 8.5. At room temperature and MgCl2 concentrations below 0.5 and above 5 mM, almost no triplex was formed. It is suggested that the absence of the triplex at higher MgCl2 concentrations is due to the stabilization of the duplex by Mg2+ ions which prevents the duplex opening necessary for the triplex formation. At higher temperatures, favorable for duplex opening (e.g. 55 degrees C), the TAT triplex is formed even in the presence of 10 mM MgCl2. Among Ca2+, Sr2+, Ba2+, Cd2+, Zn2+ and Ni2+, only Ca2+ and Sr2+ yielded a modification pattern similar to that obtained with Mg2+; the modification pattern produced in the presence of Sr2+ was, however, much less intense. In the presence of 1 mM MgCl2, a decrease in pH from 8.5 to 7.7 resulted in a strong decrease of the triplex content. At highly negative superhelix density, the conditions for triplex formation were less stringent, and the triplex was observed even in the absence of MgCl2.
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PMID:Intramolecular TAT triplex in (dA)58.(dT)58. influence of ions. 852 29

Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen. The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may potentially be involved in nickel transport. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B. bronchiseptica. We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B. bronchiseptica from phagolysosomal damage. Comparison of the urease promoter regions of B. bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B. pertussis Tohama I revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself.
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PMID:Characterisation of the urease gene cluster in Bordetella bronchiseptica. 952 76

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
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PMID:[The PTD domain of Tat protein enhance GFP protein delivering into myeloma cell SP2/0]. 1256 Dec 18

Clostridium botulinum exoenzyme C3 is responsible for the inactivation of members of the Rho GTPase family that are implicated in actin-cytoskeleton reorganization. This property has been extensively used in the field to investigate the functionality of the Rho GTPases. However, systematic analysis of Rho GTPase functions requires large amounts of such inhibitors and consequently an optimization of the production yield of these proteins. Bacterial production of soluble proteins often requires a refolding step that noticeably affects the production yields and necessitates additional experiments to verify functional activity. This is particularly true for TAT-C3, the production yields of which are generally low. In this report, we describe a rapid and efficient method for the production of soluble C3 exoenzyme developed by screening a collection of bacterial strains. The recombinant C3 protein was fused to the TAT protein-transduction domain from HIV, to allow protein delivery into cells, and to a hexahistidine tag, that permitted purification by Nickel affinity chromatography. We have demonstrated the production of large amounts of soluble and functional protein using the bacterial strain AD494 (DE3)pLysS. This rapid and efficient method for the production of soluble C3 exoenzyme could also be useful for the production of other proteins with solubility problems.
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PMID:Efficient production of Clostridium botulinum exotoxin C3 in bacteria: a screening method to optimize production yields. 1572 84

High mobility group box 1 (HMGB1) is an abundant nuclear protein that binds to double-stranded DNA. HMGB1 is composed of high mobility (HMG) box A, box B, and C-terminal acidic regions. In this study, a recombinant TAT linked HMGB1 box A (rTAT-HMGB1A) peptide was expressed, purified, and characterized as a carrier of nucleic acids. The HMGB1A cDNA was amplified by PCR, and cloned into the pET21a expression vector with the TAT domain located at the N-terminus. The rTAT-HMGB1A peptide was overexpressed and purified using Nickel affinity chromatography. A recombinant HMGB1A (rHMGB1A) peptide without the TAT domain was also overexpressed and purified as a control. In gel retardation assays, both the rHMGB1A and rTAT-HMGB1A peptides formed complexes with DNA equally well. However, transfection assays showed that the rTAT-HMGB1A peptide had a higher gene transfer efficiency than rHMGB1A. Finally, rTAT-HMGB1A had no cytotoxicity to HEK 293 cells suggesting that rTAT-HMGB1A may be useful as a non-toxic gene delivery carrier.
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PMID:Expression, purification and characterization of TAT-high mobility group box-1A peptide as a carrier of nucleic acids. 1834 54

The TAT-high mobility group box-1 A box peptide (TAT-HMGB1A) has been reported previously to be able to deliver DNA into cells without cytotoxicity. In this study, an artery wall smooth muscle cell-targeting carrier was developed using TAT-HMGB1A combined with an artery wall binding peptide (ABP). For the production of ABP linked TAT-HMGB1A (TAT-HMGB1A-ABP), pET15b-TAT-HMGB1A-ABP was constructed by inserting the ABP cDNA into pET15b-TAT-HMGB1A. TAT-HMGB1A-ABP was expressed in E. coli and purified by Nickel chelate chromatography. Gel retardation assays showed that TAT-HMGB1A-ABP formed a complex with the plasmid at or above a 5:1 weight ratio (peptide:plasmid). At a 20:1 weight ratio, the zeta-potential was approximately 25 mV and the particle size was approximately 120 nm. TAT-HMGB1A-ABP had the highest transfection efficiency in A7R5 smooth muscle cells at a weight ratio of 20:1. TAT-HMGB1A-ABP exhibited higher transfection efficiency in A7R5 cells than PLL or TAT-HMGB1A, while TAT-HMGB1A-ABP had lower transfection efficiencies in Hep3B hepatoma, 293 kidney, NIH3T3 fibroblast, and Raw264.7 macrophage cells compared with PLL. Together, these results suggest that the ABP moiety of the peptide increased transfection efficiency specifically in smooth muscle cells. In a competition assay, the transfection efficiency of TAT-HMGB1A-ABP in A7R5 cells was reduced by the addition of free ABP. MTT assays showed that TAT-HMGB1A-ABP did not produce any cytotoxicity in A7R5 cells. Therefore, TAT-HMGB1A-ABP may be useful for a targeting gene delivery to smooth muscle cells.
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PMID:A high mobility group B-1 box A peptide combined with an artery wall binding peptide targets delivery of nucleic acids to smooth muscle cells. 1928 17