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Cloning, Over-expression, and Characterization of YjgA, a Novel ppGpp-binding Protein
Gnanasekaran, Gopalsamy,Pan, SangO,Jung, Wontae,Jeong, Kwangjoon,Jeong, Jae-Ho,Rhee, Joon Haeng,Choy, Hyon E.,Jung, Che-Hun Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.8
Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.
Cloning, Over-expression, and Characterization of YjgA, a Novel ppGpp-binding Protein
Gopalsamy Gnanasekaran,,SangO Pan,Wontae Jung,정광준,정재호,이준행,최현일,Che-Hun Jung 대한화학회 2013 Bulletin of the Korean Chemical Society Vol.34 No.8
Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGppbinding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as 5.2 ± 2.0 μM, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the overexpression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.
Jian Pei,Kyung-Sub Moon,SangO Pan,Kyung-Hwa Lee,Hyang-Hwa Ryu,Tae-Young Jung,In-Young Kim,Woo-Yeol Jang,Chae-Hun Jung,Shin Jung 대한뇌종양학회 2014 Brain Tumor Research and Treatment Vol.2 No.1
Background To investigate the molecular basis for invasion of malignant gliomas, proteomic analysis approach was carried out using two human glioma cell lines, U87MG and U343MG-A that demonstrate different motility and invasiveness in in vitro experiments. Methods High-resolution two-dimensional gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry analysis were performed. Results Nine distinct protein spots that were recognized with significant alteration between the two cell lines. Five of these protein spots were up-regulated in U87MG and four were up-regulated in U343MG-A. Conclusion Among these proteins, cathepsin D was shown to be one of the important proteins which are related with glioma invasion. However, further studies are necessary to reveal the exact role and mechanism of cathepsin D in glioma invasion.