Journal Article
- [PROTOCOL] Determination of protein phosphorylation by polyacrylamide gel electrophoresis
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Chang-Ro Lee , Young-Ha Park , Huitae Min , Yeon-Ran Kim , Yeong-Jae Seok
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J. Microbiol. 2019;57(2):93-100. Published online January 31, 2019
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DOI: https://doi.org/10.1007/s12275-019-9021-y
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Abstract
- 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
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Ju-Soon Yoo , Youn-Ju Jung , Soo-Yeol Chung , Young-Choon Lee , Yong-Lark Choi
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J. Microbiol. 2004;42(3):205-210.
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DOI: https://doi.org/2088 [pii]
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Abstract
- 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.