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Tagging System (tagging + system)
Selected AbstractsTowards the high-throughput expression of metalloproteins from the Mycobacterium tuberculosis genomeJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2005John F. Hall The provision of high-quality protein in adequate quantities is a prerequisite for any structural genomics programme. A number of proteins from the Mycobacterium tuberculosis genome have been expressed and the success at each stage of the process assessed. Major difficulties have been encountered in the purification and solubilization of many of these proteins, most likely as a result of mis-folding. Some improvements have been made to the protocol but the overall success rate is still limited; however, the use of a cell-free protein expression system will circumvent some of the difficulties encountered. Alternative purification systems are also required and the properties of a mutant blue copper protein are described, that may offer a combined purification and tagging system. [source] Novel affinity tag system using structurally defined antibody-tag interaction: Application to single-step protein purificationPROTEIN SCIENCE, Issue 12 2008Terukazu Nogi Abstract Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody,peptide interaction, opening opportunities for further improvements/modifications. [source] Early events of Bacillus anthracis germination identified by time-course quantitative proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2006Pratik Jagtap Abstract Germination of Bacillus anthracis spores involves rehydration of the spore interior and rapid degradation of several of the protective layers, including the spore coat. Here, we examine the temporal changes that occur during B. anthracis spore germination using an isobaric tagging system. Over the course of 17,min from the onset of germination, the levels of at least 19 spore proteins significantly decrease. Included are acid-soluble proteins, several known and predicted coat proteins, and proteins of unknown function. Over half of these proteins are small (less than 100 amino acids) and would have been undetectable by conventional gel-based analysis. We also identified 20 proteins, whose levels modestly increased at the later time points when metabolism has likely resumed. Taken together, our data show that isobaric labeling of complex mixtures is particularly effective for temporal studies. Furthermore, we describe a rigorous statistical approach to define relevant changes that takes into account the nature of data obtained from multidimensional protein identification technology coupled with the use of isobaric tags. This study provides an expanded list of the proteins that may be involved in germination of the B. anthracis spore and their relative levels during germination. [source] Development and testing of a pedigree-marking system using visible implant elastomer tags for selective improvement in Morone breeding programmesAQUACULTURE RESEARCH, Issue 8 2010Sidney Adam Fuller Abstract The development and testing of a visible implant elastomer pedigree-marking system was evaluated in sunshine bass, Morone chrysops×Morone saxatilis, and white bass, M. chrysops (Rafinesque). These tags were tested in sunshine bass fingerlings at one of four subdermal body locations (posterior to the eye, dorsal fin musculature, caudal fin musculature or anal fin musculature). Tag visibility decreased with increased sunshine bass growth (63% after 56 days). Visibility differed among body locations, with the caudal and anal tagging locations having lower visibility. White bass fingerlings representing eight genetic groups were then tagged at one of two body locations (left or right subdermal along the dorsal musculature) using one of four fluorescent colours and reared for 42 days in a common garden growth trial. Tag visibility in white bass was 99.5% at 14 days, 98.2% at 28 days and 94.9% at 42 days after tagging. There was a significant change in weight among the eight genetic groups of white bass fingerlings after 42 days (P=0.03). Testing of this pedigree tagging system successfully identified phenotypically different groups of white bass fingerlings. [source] Evaluation of visible implant elastomer tags for tagging juvenile gilthead seabream (Sparus auratus L.); effects on growth, mortality, handling time and tag lossAQUACULTURE RESEARCH, Issue 8 2005Nicolįs Astorga Abstract The use of the fluorescent visible implant elastomer (VIE) tagging system in juvenile gilthead seabream, Sparus auratus L., between 7 and 18 g was examined. Four different colours (red, green, orange and yellow), three body positions (dorsal, lateral and caudal) and two orientations (horizontal and vertical) were tested. The mean tag application time for each fish was 15.7±0.32 s. There was no mortality associated with the method of tagging. The most visible tag colour was red. Injection orientation had a significant effect on length, width, fragmentation and fluorescent intensity of the tag. Horizontal tagging is recommended because of high fluorescent intensity, low fragmentation and for double tagging. There were no differences in growth between untagged controls and the VIE-tagged fish. [source] A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli,Expression and characterization of cytochrome-tagged Complex I subunitsPROTEIN SCIENCE, Issue 8 2010Tobias Gustavsson Abstract Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c550. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an Em of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c -tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. [source] | ||||||||||