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[PROTOCOL] Determination of protein phosphorylation by polyacrylamide gel electrophoresis
Chang-Ro Lee , Young-Ha Park , Huitae Min , Yeon-Ran Kim , Yeong-Jae Seok
J. Microbiol. 2019;57(2):93-100.   Published online January 31, 2019
DOI: https://doi.org/10.1007/s12275-019-9021-y
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AbstractAbstract
Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.

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Research Support, Non-U.S. Gov't
Molecular Cloning and Characterization of CMCase gene (celC) from Salmonella typhimurium UR
Ju-Soon Yoo , Youn-Ju Jung , Soo-Yeol Chung , Young-Choon Lee , Yong-Lark Choi
J. Microbiol. 2004;42(3):205-210.
DOI: https://doi.org/2088 [pii]
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AbstractAbstract
The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium UR1. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50^oC and pH 6.5, respectively.

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