Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

<p>Abstract</p> <p>Background</p> <p>Membrane proteins (MPs) populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as <it>E. coli </it>has curtailed progress in our understanding of their structure and function.</p> <p>Results</p> <p>Using the seven transmembrane segments <it>H. turkmenica </it>deltarhodopsin (HtdR) as a reporter, we isolated a spontaneous mutant in the arabinose-inducible <it>P</it>_BADpromoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing <it>htdR </it>transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δ<it>tig </it>cells), toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (<it>N. pharonis </it>sensory rhodopsin II) was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δ<it>tig </it>host.</p> <p>Conclusions</p> <p>Our system, which combines a downregulated version of the tightly repressed <it>P</it>_BADpromoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.</p

Protease-deficient bacterial strains for production of proteolytically sensitive polypeptides

The invention relates to the construction of protease-deficient Escherichia coli hosts which when combined with an expression system are useful for the production of proteolytically sensitive polypeptides. The invention also includes examples of particular mutant Escherichia The United States Government may have certain rights in the present invention pursuant to the terms of Grant No. CBT-8657471 awarded by the National Science Foundation.Board of Regents, University of Texas Syste

Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs

Ranking of library and information science researchers: Comparison of data sources for correlating citation data, and expert judgments

This paper studies the correlations between peer review and citation indicators when evaluating research quality in library and information science (LIS). Forty-two LIS experts provided judgments on a 5-point scale of the quality of research published by 101 scholars; the median rankings resulting from these judgments were then correlated with h-, g- and H-index values computed using three different sources of citation data: Web of Science (WoS), Scopus and Google Scholar (GS). The two variants of the basic h-index correlated more strongly with peer judgment than did the h-index itself; citation data from Scopus was more strongly correlated with the expert judgments than was data from GS, which in turn was more strongly correlated than data from WoS; correlations from a carefully cleaned version of GS data were little different from those obtained using swiftly gathered GS data; the indices from the citation databases resulted in broadly similar rankings of the LIS academics; GS disadvantaged researchers in bibliometrics compared to the other two citation database while WoS disadvantaged researchers in the more technical aspects of information retrieval; and experts from the UK and other European countries rated UK academics with higher scores than did experts from the USA. (C) 2010 Elsevier Ltd. All rights reserved

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

This is the published version. Copyright © 2012, American Society for Microbiology. All Rights Reserved.Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo

Conformational Dynamics of the Plug Domain of the SecYEG Protein-conducting Channel

The central pore of the SecYEG preprotein-conducting channel is closed at the periplasmic face of the membrane by a plug domain. To study its conformational dynamics, the plug was labeled site-specifically with an environment-sensitive fluorophore. In the presence of a stable preprotein translocation intermediate, the SecY plug showed an enhanced solvent exposure consistent with a displacement from the hydrophobic central pore region. In contrast, binding and insertion of a ribosome-bound nascent membrane protein did not alter the plug conformation. These data indicate different plug dynamics depending on the ligand bound state of the SecYEG channel.

Changing the mechanical unfolding pathway of FnIII10 by tuning the pulling strength

We investigate the mechanical unfolding of the tenth type III domain from fibronectin, FnIII10, both at constant force and at constant pulling velocity, by all-atom Monte Carlo simulations. We observe both apparent two-state unfolding and several unfolding pathways involving one of three major, mutually exclusive intermediate states. All the three major intermediates lack two of seven native beta-strands, and share a quite similar extension. The unfolding behavior is found to depend strongly on the pulling conditions. In particular, we observe large variations in the relative frequencies of occurrence for the intermediates. At low constant force or low constant velocity, all the three major intermediates occur with a significant frequency. At high constant force or high constant velocity, one of them, with the N- and C-terminal beta-strands detached, dominates over the other two. Using the extended Jarzynski equality, we also estimate the equilibrium free-energy landscape, calculated as a function of chain extension. The application of a constant pulling force leads to a free-energy profile with three major local minima. Two of these correspond to the native and fully unfolded states, respectively, whereas the third one can be associated with the major unfolding intermediates.Comment: 15 pages, 9 figure

The Hypertext Corpus Initiative:methods and tools for Social Sciences to build corpus from the web

Since its foundation in May 2009, Sciences Po’s médialab has worked to enhance the use of digital methods and tools in Social Sciences. With the help of current tools and methods, we experienced the use of web mining techniques to extract and mine digital traces (hypertext links, spontaneous expression on blogs or social networks...) of collective phenomena. Our intention is to consider the web as a field to build new kind of corpora, and not as a research object in itself (web studies), neither as a media (innovative digital mediated surveys) nor as a medium (publishing or accessing structured digital data from the web). This approach raised methodological and practical issues starting with the difficulty to build the highly accurate corpora needed by social scientists from the very complex document space that is the web : it has no size (too big, too dynamic), no clear boundaries because of its hyperlink structure and is composed of a wide heterogeneity of documents (technically, in usage, in time). How to qualitatively identify, select and collect web resources in such a quantitative context ? What does accuracy and representativity means in the moving matters of the web ? What are the tools which can equip the social scientists to build those new kind of corpora ? Because we couldn't find a good enough answer to those questions by using the existing tools we decided to launch in October 2010 the Hypertext Corpus Initiative gathering actors from web archiving, web mining, social sciences and librarians communities. HCI provides for social scientists a new set of methodology and tools, allowing them to mine more accurately digital traces of social phenomena from the web. We will present in this paper the 4 mains methodological and technical issues discussed in HCI which lead us into developing a new set of tools : (1) “what is a web corpus ?”, introducing the concept of web entities to handle the complexity and heterogeneity of web resources; (2) “how to build a web corpus ?”, the methodological and technological issues regarding the quali- quantitative process of building a web corpus proposing to organize a research driven crawling for social sciences purposes; (3) “how to analyze a web corpus ?”, we would like to identified opportunities and limitations in using the web as a research field; (4) “how to foster the use of web archives by social scientists ?”, by applying web corpus principles to the archived web