1.Characterization of Type 2 Restriction Endonucleases (Hpy51) from Helicobacter pylori Strain 51.
Myung Je CHO ; Jeong Uck PARK ; Beong Sam JEON ; Jeong Won PACK ; Eun Young BYUN ; Sun Kyung LEE ; Ye Hyoung PARK ; Jae Young SONG ; Woo Kon LEE ; Seung Chul BAIK ; Yeo Jeong CHOI ; Seun Ae JUNG ; Mi Young CHOE ; Sang Haeng CHOI ; Gyung Hyuck KO ; Hee Shang YOUN ; Kwang Ho RHEE
Journal of Bacteriology and Virology 2001;31(3):207-215
This study describes the purification and characterization of type II restriction endonuclease of Helicobacter pylori in order to understand the DNA restriction and modification of H. pylori. H. pylori cell extract was subjected to polyethyleneimine treatment, salt precipitation, heparine-sepharose column chromatography, and fast protein liquid chromatography (FPLC) using Resource Q column and Mono Q column to purify the type II restriction endonuclease. Hpy51-I was characterized to recognize the sequneces 5`-GT(G/C)AC-3`, yielding 5-base 5` protruding ends. The restriction sequence was identical to that of Tsp 45 I. The enzyme exhibited its maximal activity in the presence of 10-20 mM LaCl, but was inhibited completely in the presence of more than 80 mM NaCl. The enzyme showed its maximal activity in the presence of 1-10 mM MgC1(2). The optimal pH and temperature for enzyme activity was pH 9.0 and 37 degrees C, respectively. MnC1(2) could not substitute for MgC1(2) in reaction mixture. And addition of j3-mercaptoethanol and bovine serum albumin in reaction mixture led to loss of enzyme activity of Hpy51-I. The whole cell extract of H. pylori strain 51 was confirmed to carry the enzyme activity for methylation of Hpy51-I-recognised sequence. Hpy51-I digested genomic DNAs of enteric bacteria to less than I kb while it could not cut the genomic DNAs of H. pylori isolates. In this study, the type II restriction enzyme (Hpy51-I) of H. pylori was identified and characterized its biochemical properties, demonstrating that Hpy51-I might be one of the barriers for preventing the introduction of foreign DNAs into H. pylori.
Chromatography
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Chromatography, Liquid
;
DNA
;
DNA Restriction Enzymes*
;
Enterobacteriaceae
;
Helicobacter pylori*
;
Helicobacter*
;
Hydrogen-Ion Concentration
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Methylation
;
Polyethyleneimine
;
Serum Albumin, Bovine
2.Two-dimensional gel electrophoresis of Helicobacter pylori for proteomic analysis.
Tae Sung JUNG ; Seung Chul KANG ; Yeo Jeong CHOI ; Beong Sam JEON ; Jeong Won PARK ; Sun Ae JUNG ; Jae Young SONG ; Sang Haeng CHOI ; Seong Gyu PARK ; Mi Young CHOE ; Byung Sang LEE ; Eun Young BYUN ; Seung Chul BAIK ; Woo Kon LEE ; Myung Je CHO ; Hee Sang YOUN ; Gyung Hyuck KO ; Kwang Ho RHEE
Journal of the Korean Society for Microbiology 2000;35(2):97-108
Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer (9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)). The extract (10 mug) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.
Electrophoresis
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Electrophoresis, Gel, Two-Dimensional*
;
Helicobacter pylori*
;
Helicobacter*
;
Hydrogen-Ion Concentration
;
Molecular Weight
;
Protein Isoforms
;
Proteomics
;
Proton-Motive Force
;
Silver Staining
;
Sodium
;
Urea