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Bartholomew M Sefton - One of the best experts on this subject based on the ideXlab platform.

  • unit 13 3 Phosphoamino Acid analysis
    Current protocols in protein science, 2001
    Co-Authors: Bartholomew M Sefton
    Abstract:

    It is often valuable to identify the phosphorylated residue in a protein. This unit presents a protocol for partial Acid hydrolysis of proteins phosphorylated at serine, threonine, or tyrosine, followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid. Phosphothreonine and phosphotyrosine are more stable to hydrolysis in alkali than are RNA and phosphoserine. Therefore, an alternate procedure using mild alkaline hydrolysis of protein samples to enhance the detection of phosphothreonine and phosphotyrosine is also provided.

  • unit 18 3 Phosphoamino Acid analysis
    Current Protocols in Molecular Biology, 2001
    Co-Authors: Bartholomew M Sefton
    Abstract:

    It is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial Acid hydrolysis in HCl followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic Acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.

  • Phosphoamino Acid analysis
    Current protocols in pharmacology, 1999
    Co-Authors: Bartholomew M Sefton
    Abstract:

    : Proteins involved in signal transduction are often phosphorylated. Determination of the specific amino Acid residue(s) involved is used in characterizing the particular pathway. Partial Acid hydrolysis of phosphorylated proteins followed by two-dimensional thin layer chromatography is used to identify the phosphorylated residues of the protein as phosphoserine, phosphothreonine, or phosphotyrosine. Mild alkaline hydrolysis is used to enhance detection of phosphothreonine and phosphotyrosine.

  • Phosphoamino Acid analysis
    Current protocols in protein science, 1997
    Co-Authors: Bartholomew M Sefton
    Abstract:

    : It is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial Acid hydrolysis in HCl followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic Acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.

  • Phosphoamino Acid analysis
    Current protocols in protein science, 1997
    Co-Authors: Bartholomew M Sefton
    Abstract:

    It is often valuable to identify the phosphorylated residue in a protein. This unit presents a protocol for partial Acid hydrolysis of proteins phosphorylated at serine, threonine, or tyrosine, followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid. Phosphothreonine and phosphotyrosine are more stable to hydrolysis in alkali than are RNA and phosphoserine. Therefore, an alternate procedure using mild alkaline hydrolysis of protein samples to enhance the detection of phosphothreonine and phosphotyrosine is also provided.

Tony Hunter - One of the best experts on this subject based on the ideXlab platform.

  • phosphopeptide mapping and Phosphoamino Acid analysis on cellulose thin layer plates
    Cell Biology#R##N#A Laboratory Handbook Volume 3, 1994
    Co-Authors: Peter Van Der Geer, Bartholomew M Sefton, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the use of phosphopeptide mapping and Phosphoamino Acid analysis on thin-layer cellulose. Phosphopeptide mapping is an important technique in the study of protein phosphorylation. It is used to determine the number and precise identity of sites of phosphorylation, to estimate the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for their phosphorylation. Additionally, comparative phosphopeptide mapping is an invaluable tool for determining the identity, or lack of identity of phosphoproteins obtainable only in trace amounts. Phosphopeptide mapping has the advantage that it is extremely sensitive. In addition, individual phosphopeptides can be isolated from the inert cellulose coating of the plate, and used for further characterization including Phosphoamino Acid determination, N-terminal sequencing, and secondary digestion with additional proteases and chemicals.

  • phosphopeptide mapping and Phosphoamino Acid analysis by electrophoresis and chromatography on thin layer cellulose plates
    Electrophoresis, 1994
    Co-Authors: Peter Van Der Geer, Tony Hunter
    Abstract:

    : Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the Phosphoamino Acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial Acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

  • phosphopeptide mapping and Phosphoamino Acid analysis by two dimensional separation on thin layer cellulose plates
    Methods in Enzymology, 1991
    Co-Authors: William J Boyle, Peter Van Der Geer, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the phosphopeptide mapping and Phosphoamino Acid analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino Acid composition and sequence, and (3) to determine the precise location of amino Acid residues that are posttranslationally modified by fatty Acid acylation, glycosylation, methylation, acetylation, or phosphorylation.

Jesse Rinehart - One of the best experts on this subject based on the ideXlab platform.

  • Expanding the Genetic Code of Escherichia coli with Phosphoserine
    2016
    Co-Authors: Jack Benner, Christopher J Noren, Jesse Rinehart, Dieter Söll
    Abstract:

    O-Phosphoserine (Sep), the most abundant Phosphoamino Acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular biology, and disease research. O-Phosphoserine (Sep) was identified 80years ago as a constituent of phospho-proteins from egg yolk (1). Since then, the extent and importance of the eukaryotic phos-phoproteome has been realized and has provided insight into large interconnected networks of kinases and phosphatases (2). Protein kinase

  • Expanded cellular amino Acid pools containing phosphoserine, phosphothreonine, and phosphotyrosine
    2016
    Co-Authors: Justin B Steinfeld, Hans Rudolf Aerni, Svetlana Rogulina, Yuchen Liu, Jesse Rinehart
    Abstract:

    ABSTRACT: Adding nonstandard amino Acids to the genetic code of E. coli expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino Acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accom-plished, conditions that modulate intracellular Phosphoamino Acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in E. coli. We show that deletion of the phosphoserine phosphatase serB elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino Acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were grown in LB with supplemented phosphothreonine and ser

  • Expanded Cellular Amino Acid Pools Containing Phosphoserine, Phosphothreonine, and Phosphotyrosine
    2015
    Co-Authors: Justin B. Steinfeld, Hans Rudolf Aerni, Svetlana Rogulina, Yuchen Liu, Jesse Rinehart
    Abstract:

    Adding nonstandard amino Acids to the genetic code of E. coli expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino Acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accomplished, conditions that modulate intracellular Phosphoamino Acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in E. coli. We show that deletion of the phosphoserine phosphatase serB elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino Acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were grown in LB with supplemented phosphothreonine and serB deficient cells were grown in low phosphate media with supplemented phosphotyrosine, respectively. These findings remove a major barrier for further expansion of the genetic code with additional phosphorylated amino Acids

  • expanded cellular amino Acid pools containing phosphoserine phosphothreonine and phosphotyrosine
    ACS Chemical Biology, 2014
    Co-Authors: Justin B Steinfeld, Hans Rudolf Aerni, Svetlana Rogulina, Jesse Rinehart
    Abstract:

    Adding nonstandard amino Acids to the genetic code of E. coli expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino Acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accomplished, conditions that modulate intracellular Phosphoamino Acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in E. coli. We show that deletion of the phosphoserine phosphatase serB elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino Acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were gr...

  • expanding the genetic code of escherichia coli with phosphoserine
    Science, 2011
    Co-Authors: Heesung Park, Edith Osborne, Jack S Benner, Christopher J Noren, Michael J Hohn, Jesse Rinehart, Takuya Umehara
    Abstract:

    O -Phosphoserine (Sep), the most abundant Phosphoamino Acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA Sep ), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA Sep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep 218 , Sep 222 ). This system has general utility in protein engineering, molecular biology, and disease research.

Shouyi Chen - One of the best experts on this subject based on the ideXlab platform.

  • evidence for serine threonine and histidine kinase activity in the tobacco ethylene receptor protein nthk2
    Plant Physiology, 2004
    Co-Authors: Zhigang Zhang, Hualin Zhou, Tao Chen, Yan Gong, Wanhong Cao, Yujun Wang, Jinsong Zhang, Shouyi Chen
    Abstract:

    Ethylene plays important roles in plant growth, development, and stress responses. Two ethylene receptors, ETR1 from Arabidopsis and NTHK1 from tobacco (Nicotiana tabacum), have been found to have His kinase (HK) activity and Ser/Thr kinase activity, respectively, although both show similarity to bacterial two-component HK. Here, we report the characterization of another ethylene receptor homolog gene, NTHK2, from tobacco. This gene also encodes a HK-like protein and is induced by dehydration and CaCl(2) but not significantly affected by NaCl and abscisic Acid treatments. The biochemical properties of the yeast (Schizosaccharomyces pombe)-expressed NTHK2 domains were further characterized. We found that NTHK2 possessed Ser/Thr kinase activity in the presence of Mn(2+) and had HK activity in the presence of Ca(2+). Several lines of evidence supported this conclusion, including hydrolytic stability, Phosphoamino Acid analysis, mutation, deletion, and substrate analysis. These properties have implications in elucidation of the complexity of the ethylene signal transduction pathway and understanding of ethylene functions in plants.

  • serine threonine kinase activity in the putative histidine kinase like ethylene receptor nthk1 from tobacco
    Plant Journal, 2003
    Co-Authors: Can Xie, Zhigang Zhang, Hualin Zhou, Jinsong Zhang, Daowen Wang, Shouyi Chen
    Abstract:

    A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing Acid/base stability, Phosphoamino Acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.

Peter Van Der Geer - One of the best experts on this subject based on the ideXlab platform.

  • phosphopeptide mapping and Phosphoamino Acid analysis on cellulose thin layer plates
    Cell Biology#R##N#A Laboratory Handbook Volume 3, 1994
    Co-Authors: Peter Van Der Geer, Bartholomew M Sefton, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the use of phosphopeptide mapping and Phosphoamino Acid analysis on thin-layer cellulose. Phosphopeptide mapping is an important technique in the study of protein phosphorylation. It is used to determine the number and precise identity of sites of phosphorylation, to estimate the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for their phosphorylation. Additionally, comparative phosphopeptide mapping is an invaluable tool for determining the identity, or lack of identity of phosphoproteins obtainable only in trace amounts. Phosphopeptide mapping has the advantage that it is extremely sensitive. In addition, individual phosphopeptides can be isolated from the inert cellulose coating of the plate, and used for further characterization including Phosphoamino Acid determination, N-terminal sequencing, and secondary digestion with additional proteases and chemicals.

  • phosphopeptide mapping and Phosphoamino Acid analysis by electrophoresis and chromatography on thin layer cellulose plates
    Electrophoresis, 1994
    Co-Authors: Peter Van Der Geer, Tony Hunter
    Abstract:

    : Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the Phosphoamino Acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial Acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

  • phosphopeptide mapping and Phosphoamino Acid analysis by two dimensional separation on thin layer cellulose plates
    Methods in Enzymology, 1991
    Co-Authors: William J Boyle, Peter Van Der Geer, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the phosphopeptide mapping and Phosphoamino Acid analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino Acid composition and sequence, and (3) to determine the precise location of amino Acid residues that are posttranslationally modified by fatty Acid acylation, glycosylation, methylation, acetylation, or phosphorylation.