KMID : 0613820180280010099
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Journal of Life Science 2018 Volume.28 No. 1 p.99 ~ p.104
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C-terminal Fusion of EGFP to Pneumolysin from Streptococcus pneumoniae modified its Hemolytic Activity
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Chung Kyung-Tae
Lee Jae-Heon Jo Hye-Ju
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Abstract
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Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by Ni2+ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3¡® end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate?polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY¡¯s C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.
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KEYWORD
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Bacterial toxin, cytolysin, hemolysis, pneumolysin, Streptococcus pneumoniae
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