Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.     

The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.     

Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC

The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.     

Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.     

The sensor kinase CitA (DpiB) of Escherichia coli functions as a high-affinity citrate receptor

For the CitA-CitB (DpiB-DpiA) two-component signal transduction system from Escherichia coli, three diverse functions have been reported: induction of the citrate fermentation genes citCDEFXGT, repression of the regulator gene appY, and destabilization of the inheritance of iteron-containing plasmids such as pSC101. This poses the question of the principal biological role of this system. Here it is shown that the periplasmic domain of the E. coli sensor kinase CitA functions as a high-affinity citrate receptor. Two CitA derivatives were purified by affinity chromatography and subjected to binding studies using isothermal titration calorimetry (ITC). One of them, termed CitA215MBP, comprised the N-terminal part of CitA (amino acid residues 1-215), including the two transmembrane helices, and was fused to the amino terminus of the E. coli maltose-binding protein lacking its signal peptide. The second CitA derivative, designated CitAP(Ec), encompassed only the periplasmic domain (amino acid residues 38-177). CitA215MBP bound citrate at 25 degrees C with a K(d) of 0.3 microM and a binding stoichiometry of up to 0.9 in 50 mM sodium phosphate buffer, pH 7. Binding was driven by the enthalpy change (Delta H of -95.7 kJ mol(-1)), whereas the entropy change was not favorable for binding ( T Delta S of -58.6 kJ mol(-1)). ITC experiments with CitAP(Ec) yielded similar K(d) values for citrate (0.15-1.0 microM). Besides citrate, also isocitrate ( K(d) approximately tricarballylate ( K(d) approximately t not malate were bound by CitAP(Ec). The results favor the assumption that the primary biological function of the CitA-CitB system is the regulation of the citrate fermentation genes.     

In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2+

With increasing addition of Escherichia coli LPS to calf thymus DNA, both dissolved in CaCl2, absorption maxima of DNA at 260 nm decreased gradually with the appearance of isosbastic points at both ends of spectra, which implied some binding between DNA and LPS. Hill plot of absorbance data showed that the binding interaction was positive cooperative in nature. For any fixed concentration of DNA and LPS, extent of interaction increased as concentration of CaCl2 was raised from 1.0 to 100 mM, signifying the electrostatic nature of the interaction, mediated through Ca2+ ion. Stepwise addition of EDTA, a chelating agent for divalent cations, to DNA-LPS bound complex gradually reversed the spectral shift with increase in absorbance at 260 nm, which implied opening up of the complex, that is, reversible nature of the interaction. Circular dichroism spectral changes of DNA by the addition of LPS indicated partial transition of DNA from B to A form. Isothermal titration calorimetric (ITC) study showed that the DNA-LPS binding was an exothermic and enthalpy-driven phenomenon. Moreover, in the presence of 100 mM CaCl2, binding constant of the interaction was found to be 2.6 x 10(4) M(-1) and 3.1 x 10(4) M(-1) from the analysis of Hill plot and ITC result, respectively. DNA-melting study showed that the LPS binding had increased the melting temperature of DNA, indicating more stabilization of DNA double helix. The binding of LPS to DNA made the complex resistant to digestion with endonucleases EcoRI and DNase I.     

Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.     

Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.     

黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 kD,对革兰氏阳性菌(G+)和革兰氏阴性菌(G?)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体pET22b-ELP20-EK-YFGAP、pET22b-ELP40-EK-YFGAP和pET22b-ELP40-L-EK- YFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。     

BdcA,a Protein Important for Escherichia coli Biofilm Dispersal,Is a Short-Chain Dehydrogenase/Reductase that Binds Specifically to NADPH

The Escherichia coli protein BdcA (previously referred to as YjgI) plays a key role in the dispersal of cells from bacterial biofilms, and its constitutive activation provides an attractive therapeutic target for dismantling these communities. In order to investigate the function of BdcA at a molecular level, we integrated structural and functional studies. Our 2.05 Å structure of BdcA shows that it is a member of the NAD(P)(H)-dependent short-chain dehydrogenase/reductase (SDR) superfamily. Structural comparisons with other members of the SDR family suggested that BdcA binds NADP(H). This was demonstrated experimentally using thermal denaturation studies, which showed that BcdA binds specifically to NADPH. Subsequent ITC experiments further confirmed this result and reported a K_d of 25.9 µM. Thus, BdcA represents the newest member of the limited number of oxidoreductases shown to be involved in quorum sensing and biofilm dispersal.     

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

Rat liver fatty acid binding protein (L-FABP) was efficiently expressed in Escherichia coli and purified to homogeneity. The cDNA encoding L-FABP was ligated into the pTrc99A expression vector and expressed by induction with isopropyl-beta-d-thiogalactopyranoside under the control of the P(trc) promoter. Following an 18 h induction period, L-FABP constituted approximately 3% of the cytosolic protein. The protein could be purified to electrophoretic homogeneity (silver-stained polyacrylamide gel detection) by ammonium sulfate fractionation (65% saturation) of the soluble bacterial lysate followed by the chromatographic sequence of anion-exchange-->hydrophobic interaction-->anion-exchange chromatography. The recombinant protein displayed an isoelectric point of 7.0 and cross-reactivity with rabbit anti-(human L-FABP) polyclonal antibody. The ligand binding properties of the delipidated L-FABP were examined by titration with the fluorescent probe 1-anilino-8-naphthalene sulfonic acid and isothermal titration calorimetric analysis of oleic acid binding. The purified rat L-FABP displayed a binding stoichiometry of 2:1 (ANS:L-FABP) with dissociation constants (K(d)) of 1.7 and 15.5 microM for the high and low affinity binding sites, respectively. The K(d) values determined by ITC for oleic acid binding were 0.155 and 4.04 microM with a binding stoichiometry of approximately 2 mol of fatty acid/mol of protein. These physicochemical and binding properties are in agreement with those of L-FABP isolated from rat liver tissue.     

Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in k(cat). The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction. The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

Pathogenic Escherichia coli

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.     

Phospholipases of Escherichia coli

Phospholipase A activity was hardly detected in Escherichia coli K12 sonicate when solvent-extracted (free) ³²P-phosphatides were used as substrate. Phosphatides bound in membrane were, however, actively hydrolyzed to give the corresponding lysolipids by an endogenous enzyme. The results indicated the presence in E. coli membrane of a novel phospholipase which can be more precisely called as lipoprotein phospholipase A. Lysophospholipase was shown to be present in the cellular soluble fraction.

With free phosphatides as substrate, alcohols and some water-miscible solvents, as well as nonionic detergents, markedly stimulated phospholipase A activity of the membrane, possibly by enabling the substrate to hold physical state in someway simillar to that in the membrane. Possible role of this enzyme in membrane lipid metabolism is discussed.     

Mammary pathogenic Escherichia coli

Pathogenic Escherichia coli can be classified into several pathotypes, and it is believed that each pathotype carries one or more specific gene repertoire (or virulence factors combination) that distinguishes them from non-pathogenic E. coli strains and from other pathotypes. In contrast to this notion, it was proposed that this is not the case for E. coli mastitis, a common disease in farm animals and that any given E. coli isolate can cause this disease, even strains that are considered non-pathogenic. In this review we will re-examine this latter concept and recent advances in the study E. coli mastitis.