Protein synthesis in Escherichia coli at 4 degrees C

Two-dimensional electrophoresis technology was used to investigate protein synthesis by the mesophilic bacterium Escherichia coli at low temperature. It was confirmed that protein synthesis in E. coli decreased strongly after a temperature downshift from 37 to 4 degrees C. After incubation for 150 min at 4 degrees C, however, the number of synthesized proteins represented 60% of the overall polypeptide number observed at 37 degrees C. Furthermore, the analysis of autoradiograms revealed the overexpression of 69 proteins by shocked bacteria, showing that the number of cold-induced proteins has been significantly underestimated so far.     

Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hy-drophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coli. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hy-drophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni2. agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coil cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.     

Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms

The two-dimensional electrophoretic patterns of cellular proteins from gel-entrapped Escherichia coli cells were compared to those of exponential- and stationary-phase free-floating organisms. The amounts of several proteins in immobilized cells were significantly different from those in free bacteria. Immobilized organisms rapidly produced a high level of dipeptide permease and a single-strand binding protein, and progressively accumulated an aldehyde dehydrogenase. Immobilization also induced a decrease in the levels of two proteins, i.e., the YFID protein and a DNA-binding, stationary-phase protein. The possible role of these proteins in the high resistance of immobilized bacteria to stresses is discussed.     

Protein photocrosslinking reveals dimer of dimers formation on MarR protein in Escherichia coli

The multiple antibiotic resistance regulatory protein(MarR) binds to two promoter sites on the marO operator in Escherichia coli.Our study showed that more than one MarR dimer proteins bound to either of its two promoter sites(Site I and Site II),suggesting that MarR might form higher complexes than homodimers when bound to DNA inside E.coli cells.To further verify this hypothesis,we site-specifically incorporated a photocrosslinking probe at the interface between two MarR dimer proteins.Photolysis in living E.coli cells revealed a covalent linkage between the two interdimer subunits of MarR,suggesting that MarR forms dimer of dimers in vivo.     

过氧亚硝酸根对蛋白质损伤的研究进展

过氧亚硝酸根--一氧化氮和超氧根阴离子快速结合的产物,是生物体内产生的一种强氧化剂和细胞毒性物质,它可以介导生物大分子包括DNA、蛋白质和脂质的修饰和损伤。近年来,由于许多疾病条件下都发现大量过氧亚硝酸根诱导的蛋白质损伤产物,因此过氧亚硝酸根与蛋白质的相互作用引起了人们的广泛关注。本文详尽地介绍了过氧亚硝酸根介导蛋白质损伤对人体的病理作用和过氧亚硝酸根与蛋白质作用机制等方面的最新研究进展,并对未来过氧亚硝酸根与蛋白质相互作用的研究作了展望。     

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)₇₀, one molecule of (dT)₃₅, or two molecules of (dT)₃₅] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.      

Recombinant Production of Crab Antimicrobial Protein Scygonadin Expressed as Thioredoxin and SUMO Fusions in Escherichia coli

Scygonadin is an antimicrobial protein isolated from the mud crab, Scylla serrate. The mature protein comprises 102 amino acids and has a theoretical molecular weight of 11,272 Da. The protein’s specific expression pattern strongly suggests that it plays a role in reproductive immunity. In this study, I developed a protocol for producing recombinant scygonadin in Escherichia coli. The target protein was expressed as both thioredoxin and SUMO fusions, and released by TEV and SUMO protease-mediated cleavages, respectively. In either case, the liberated scygonadin was separated from its carrier using a HisTrap HP column. From thioredoxin and SUMO fusion constructs, 32.7 and 29.2 mg target protein per liter of culture was obtained, respectively. The described protocol provides an effective means for producing scygonadin in relatively large quantities, which facilities its further characterization.     

Sol-Gel Entrapment of Escherichia coli

Whole cell bacteria have been entrapped within sol-gel silica matrices in order to perform bio-catalytic experiments. Escherichia coli have been chosen as a model for sol-gel encapsulation. Transmission electron microscopy shows that bacteria are randomly dispersed within the silica matrix and that their cellular organization is preserved. The -galactosidase activity of entrapped E. coli was studied using p-NPG as a substrate. The formation of p-nitrophenol was followed by optical absorption. These experiments show that E. coli still exhibit noticeable enzymatic activity after encapsulation in wet gels. They follow the well known Michaelis-Menten kinetic law but their activity decreases in dried xerogels.     

Biotransformation of Benzoate to 2,4,6-Trihydroxybenzophenone by Engineered Escherichia coli

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.     

Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli

We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.     

PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from Escherichia coli.

Distance fingerprinting : Pulsed electron–electron double resonance spectroscopy (PELDOR) is applied to the octameric membrane protein complex Wza of E. coli. The data yielded a detailed distance fingerprint of its periplasmic region that compares favorably to the crystal structure. These results provide the foundation to study conformation changes from interaction with partner proteins.

     

Biological Removal of Phosphate at Low Concentrations Using Recombinant Escherichia coli Expressing Phosphate-Binding Protein in Periplasmic Space

During wastewater treatment, phosphate removal is an important and challenging process; thus, diverse technologies, including those derived from biological means, have been devised for efficient phosphate removal. Although conventional biological methods are effective in decreasing wastewater phosphate levels to ~1 mg/L, long periods of microbial adaptation are required for effective phosphate removal, and the removal efficiency of these methods is relatively poor at lower phosphate concentrations. In the present work, we constructed a recombinant Escherichia coli with periplasmic-expressed phosphate-binding protein (PBP) and investigated its biological removal ability for low phosphate levels. We found that the PBP-expressing recombinant E. coli cells showed efficient (> 94 %) removal of phosphate at low concentrations (0.2–1.0 mg/L) in a treated cell mass-dependent manner. Collectively, we propose that our PBP-expressing recombinant whole-cell system could be successfully used during wastewater treatment for the biological removal of low concentrations of phosphate.     

Rapid methods to enumerate Escherichia coli in foods using 4-methylumbelliferyl-beta-D-glucuronide

Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-beta-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)-MUG medium. An efficacious and cost-effective method is needed that can detect E. coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. coli and other lactose-fermenting organisms, the proposed method using regular LST/EC-MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC-MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC-MUG medium. For a product with a history of being positive for E. coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST-MUG is the method of choice. A presumptive positive in the LST-MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. coli. For samples spiked with E. coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. coli.     

银纳米粒子对大肠杆菌和金黄葡萄球菌杀灭机理研究

本文对银纳米粒子的杀菌活性和杀菌机理进行了初步探讨.实验表明,400 μL的0.2 mmol·L-1银纳米粒子加入到1 mL自制的大肠杆菌溶液(600个/毫升)中,就具有明显的杀菌效果.银纳米粒子浓度增加,杀菌时间延长,杀菌效果明显增强;温度对杀菌效果有明显的影响,在温度达到3℃以后,杀菌效果显著,在低于这个温度时,银...     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.