Hyogyung Shin. (2023). Characterization of Endospore Proteins Associated with DNA Protection. doi: 10.22677/THESIS.200000686904
Type
Thesis
Description
Clostridium perfringens; endospore; small acid-soluble protein; DNA protection; sortase-mediated fluorescent labeling
Table Of Contents
Ⅰ. Introduction 1 Ⅱ. Materials and Methods 9 2.1 Wild-type Ssp4 expression plasmids 9 2.2 Purification of Ssp4 9 2.3 Preparation of DNA 10 2.4 Electrophoretic mobility shift assay (EMSA) 11 2.5 DNA protection assay against UV irradiation 12 2.6 DNA protection assay against DNase I degradation 13 2.7 Site-directed mutagenesis for N-terminal GGG-Ssp4 14 2.8 Purification of sortase A 15 Ⅲ. Results 17 3.1 Purification of Ssp4:D36 and Ssp4:G36 proteins 17 3.2 DNA binding affinity for Ssp4 variants for random-sequence DNA 20 3.3 DNA binding affinity for Ssp4 variants for AT-rich DNA 24 3.4 DNA protection activity of Ssp4 variants 27 3.5 Sortase-mediated fluorescent labeling 31 3.5.1 Construction of N-terminal GGG-Ssp4 vector 31 3.5.2 Purification of sortase A 34 3.5.3 Establishment of sortase-mediated fluorescent labeling strategy for Ssp4 36 Ⅳ. Discussion 40 References 44 Summary in Korean 56
