Overexpression, purification and characterization of the Escherichia coli MelR transcription activator protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Caswell R
PROVIDER: S-EPMC1133192 | biostudies-other | 1992 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Objectives:To purify and characterize the glutathione binding protein GsiB of glutathione importer (GSI) in Escherichia coli (E. coli). Results:The coding sequence of GsiB was cloned from E. coli MG1655 and expressed in BL21(DE3). GsiB protein was expressed and purified to homogeneity using Ni-affinity and gel filtration chromatography. SDS-PAGE of purified GsiB showed a single protein band of molecular mass 56 kDa, while native gel showed two bands around 56 kDa and 110 kDa. Gene knockout showed that GsiB was essential for GSI mediated glutathione import. Interactions of GsiA, B, C, and D were determined using bacterial two-hybrid method. Without glutathione, GsiB showed no direct interaction with the other three proteins. However, GsiB could interact with GsiC and GsiD when using glutathione as sole sulfur source. Conclusions:GsiB functions in E. coli was characterized which could help elucidate the glutathione import mechanism in gram-negative bacteria.
| S-EPMC6236770 | biostudies-literature
Project description:Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.
| S-EPMC1839922 | biostudies-literature
Project description:We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.
| S-EPMC1317337 | biostudies-literature
Project description:Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate-responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high-level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single-chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.
| S-EPMC9314961 | biostudies-literature
Project description:A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
| S-EPMC2567230 | biostudies-literature
Project description:MelR is an Escherichia coli transcription factor belonging to the AraC family. It activates expression of the melAB operon in response to melibiose. Full-length MelR (MelR303) binds to two pairs of sites upstream of the melAB transcription start site, denoted sites 1' and 1 and sites 2 and 2', and to a fifth site, R, which overlaps the divergent melR promoter. The C-terminal domain of MelR (MelR173) does not activate transcription. Here we show that, like MelR303, when MelR173 binds to sites 1 and 2 it recruits CRP to bind between these sites. Hence, the C-terminal domain is involved in heterologous interactions. MelR173 binds to the R site, which has 11 of 18 bp identical to sites 1 and 2 but, surprisingly, does not bind to site 1', which has 12 of 18 bp identical, nor to site 2'. Using electrophoretic mobility shift assays, we show that the binding of MelR303 to sites 1' and 2' is due to cooperative binding with the adjacent site. This homologous cooperativity requires the N-terminal domain of the protein. Activation of the melAB promoter requires MelR to occupy site 2', which overlaps the -35 hexamer. Hence, both domains of MelR are required for transcription activation.
| S-EPMC117283 | biostudies-literature
Project description:BackgroundThe gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue.ResultsIn this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin.ConclusionsGg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.
| S-EPMC4234410 | biostudies-literature
Project description:Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2,3-diacylglucosamine 1-phosphate --> 2',3'-diacylglucosamine-(beta,1'-6)-2,3-diacylglucosamine 1-phosphate + UDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consists entirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purified E. coli and Haemophilus influenzae LpxB to near homogeneity on a 10-100 mg scale using protease-cleavable His(10)-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E. coli LpxB (M(r) approximately 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized with buffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide. Transmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxB is overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which no residual catalytic activity was detected. Our results provide a rational starting point for structural studies.
| S-EPMC2787792 | biostudies-literature
Project description:A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.
| S-EPMC1148925 | biostudies-other
Project description:Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app=(5.2±0.20)×10(2)M(-1)s(-1), and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×10(5)M(-1)s(-1). Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.
| S-EPMC3985065 | biostudies-literature