The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Jason P. Gallivan - One of the best experts on this subject based on the ideXlab platform.
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Genetic screens and Selections for small molecules based on a synthetic riboswitch that activates protein translation
Journal of the American Chemical Society, 2004Co-Authors: Shawn K. Desai, Jason P. GallivanAbstract:Genetic Selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of Genetic Selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to Genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing Genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive Genetic screens and Selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo Genetic Selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use Genetic Selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.
Lance H. Baumgard - One of the best experts on this subject based on the ideXlab platform.
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Effects of Week of Lactation and Genetic Selection for Milk Yield on Milk Fatty Acid Composition in Holstein Cows
Journal of Dairy Science, 2010Co-Authors: J.k. Kay, W.j. Weber, C.e. Moore, B.a. Crooker, Dale E. Bauman, L.b. Hansen, Hugh Chester-jones, Lance H. BaumgardAbstract:Control (CL) and select line (SL) dairy cows (n = 22) managed identically but differing in milk yield (>4100 kg/305 d) were used to determine differences in milk fatty acid profile as lactation progressed. Milk yield was recorded daily and milk samples were collected during wk 1, 4, 8, 12, and 16 postpartum for milk composition analysis. Milk samples from wk 1, 8, and 16 were also analyzed for fatty acid composition. Select-line cows produced more milk (44.4 vs. 31.2 kg/d) and milk components than CL cows during the 16-wk period. There was no difference in rate of milk yield increase, but peak milk yield for SL cows was greater and occurred later in lactation. There were no differences in milk SCC or milk fat, protein, or lactose content. Selection for milk yield did not affect the content of most individual milk fatty acids; however, compared with CL, SL cows had a reduced Delta(9)-desaturase system and tended to produce milk with lower monounsaturated fatty acid content. Selection for milk yield did not affect milk fatty acid origin but the percentage of de novo fatty acids increased and preformed fatty acids decreased as lactation progressed. Milk fat trans-11 18:1 and cis-9,trans-11 conjugated linoleic acid increased with progressing lactation (10.7 vs. 14.1 and 3.1 vs. 5.4 mg/g, or 31 and 76%, respectively) and were correlated strongly among wk 1, 8, and 16 of lactation. Temporal changes in the Delta(9)-desaturase system occurred during lactation but these changes were not correlated with milk fat cis-9,trans-11 conjugated linoleic acid content. Results indicate prolonged Genetic Selection for milk yield had little effect on milk fatty acid composition, but milk fatty acid profiles varied markedly by week of lactation.
Shawn K. Desai - One of the best experts on this subject based on the ideXlab platform.
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Genetic screens and Selections for small molecules based on a synthetic riboswitch that activates protein translation
Journal of the American Chemical Society, 2004Co-Authors: Shawn K. Desai, Jason P. GallivanAbstract:Genetic Selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of Genetic Selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to Genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing Genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive Genetic screens and Selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo Genetic Selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use Genetic Selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.
J.k. Kay - One of the best experts on this subject based on the ideXlab platform.
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Effects of Week of Lactation and Genetic Selection for Milk Yield on Milk Fatty Acid Composition in Holstein Cows
Journal of Dairy Science, 2010Co-Authors: J.k. Kay, W.j. Weber, C.e. Moore, B.a. Crooker, Dale E. Bauman, L.b. Hansen, Hugh Chester-jones, Lance H. BaumgardAbstract:Control (CL) and select line (SL) dairy cows (n = 22) managed identically but differing in milk yield (>4100 kg/305 d) were used to determine differences in milk fatty acid profile as lactation progressed. Milk yield was recorded daily and milk samples were collected during wk 1, 4, 8, 12, and 16 postpartum for milk composition analysis. Milk samples from wk 1, 8, and 16 were also analyzed for fatty acid composition. Select-line cows produced more milk (44.4 vs. 31.2 kg/d) and milk components than CL cows during the 16-wk period. There was no difference in rate of milk yield increase, but peak milk yield for SL cows was greater and occurred later in lactation. There were no differences in milk SCC or milk fat, protein, or lactose content. Selection for milk yield did not affect the content of most individual milk fatty acids; however, compared with CL, SL cows had a reduced Delta(9)-desaturase system and tended to produce milk with lower monounsaturated fatty acid content. Selection for milk yield did not affect milk fatty acid origin but the percentage of de novo fatty acids increased and preformed fatty acids decreased as lactation progressed. Milk fat trans-11 18:1 and cis-9,trans-11 conjugated linoleic acid increased with progressing lactation (10.7 vs. 14.1 and 3.1 vs. 5.4 mg/g, or 31 and 76%, respectively) and were correlated strongly among wk 1, 8, and 16 of lactation. Temporal changes in the Delta(9)-desaturase system occurred during lactation but these changes were not correlated with milk fat cis-9,trans-11 conjugated linoleic acid content. Results indicate prolonged Genetic Selection for milk yield had little effect on milk fatty acid composition, but milk fatty acid profiles varied markedly by week of lactation.
Matthew P. Delisa - One of the best experts on this subject based on the ideXlab platform.
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A survival Selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins
Nature Communications, 2019Co-Authors: Bunyarit Meksiriporn, Morgan B. Ludwicki, Erin A. Stephens, Allen Jiang, Dujduan Waraho-zhmayev, Lutz Kummer, Fabian Brandl, Hyeon-cheol Lee, Andreas Plückthun, Matthew P. DelisaAbstract:There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a Genetic Selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). We then use the Selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. Collectively, these results establish our Genetic Selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity.Protein phosphorylation helps to control many important cellular activities. Here the authors describe a Genetic Selection strategy to isolate designed ankyrin repeat proteins that bind specifically to phosphomodified targets.
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Engineered Genetic Selection links in vivo protein folding and stability with asparagine-linked glycosylation
Biotechnology Journal, 2013Co-Authors: Thomas J. Mansell, Cassandra Guarino, Matthew P. DelisaAbstract:Predicting the structural consequences of site-specific glycosylation remains a major challenge due in part to the lack of convenient experimental tools for rapidly determining how glycosylation influences protein folding. To address this shortcoming, we developed a Genetic Selection that directly links the in vivo folding of asparagine-linked (N-linked) glycoproteins with antibiotic resistance. Using this assay, we identified three known or putative glycoproteins from Campylobacter jejuni (Peb3, CjaA, and Cj0610c) whose folding was significantly affected by N-glycosylation. We also used the Genetic Selection to isolate a glycoengineered variant of the Escherichia coli colicin E7 immunity protein (Im7) whose intracellular folding and stability were enhanced as a result of N-glycosylation. In addition to monitoring the effect of glycan attachment on protein folding in living cells, this strategy could easily be extended for optimizing protein folding in vivo and engineering glycosylation enzymes, pathways, and hosts for optimal performance. See accompanying commentary by Danielle Tullman-Ercek DOI: 10.1002/biot.201300319
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Identifying and optimizing intracellular protein-protein interactions using bacterial Genetic Selection.
Methods of Molecular Biology, 2011Co-Authors: Dujduan Waraho, Matthew P. DelisaAbstract:Protein-protein interactions are crucial for the vast majority of biological processes. To fully understand these processes therefore requires methods for identifying protein interactions within the complex cellular environment. To isolate interacting proteins, we have developed a simple and reliable Genetic Selection by exploiting the inbuilt "hitchhiker" mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method is based on the unique ability of the Tat system to efficiently co-localize noncovalent complexes of two folded polypeptides to the periplasmic space of E. coli. The Genetic Selection is comprised of two engineered fusion proteins: an N-terminal Tat signal peptide fused to the protein of interest, and the known or putative partner protein fused to mature TEM-1 β-lactamase. The efficiency with which co-localized β-lactamase chimeras are exported in the periplasm, and thus confer ampicillin resistance to cells, is directly linked to the relative binding affinity of the protein-ligand system. Thus, ampicillin resistance can be used as a convenient readout for identifying and optimizing protein interactions in E. coli. Furthermore, because Tat substrates must be correctly folded for export, our method favors the identification of soluble, non-aggregating, protease-resistant protein pairs. Overall, we anticipate that this new Selection tool will be useful for discovering and engineering protein drugs, protein complexes for structural biology, factors that inhibit PPIs, and components for synthetic biology.