Type of Document Dissertation Author Hardt, Markus Author's Email Address firstname.lastname@example.org URN etd-0807102-094558 Title Study of a Bacillus circulans Chitin-Binding Domain by a Green Fluorescent Protein Binding Assay and Detection of Lysozymes by Improved Zymograms Degree Doctor of Philosophy (Ph.D.) Department Biochemistry (Biological Sciences) Advisory Committee
Advisor Name Title Roger A Laine Committee Chair Ding S Shih Committee Member Grover L Waldrop Committee Member Sue G Bartlett Committee Member Frank K Cartledge Dean's Representative Keywords
- green fluorescent protein
- binding assay
- affinity electrophoresis
Date of Defense 2002-07-23 Availability unrestricted AbstractA fluorescent binding assay was developed to investigate the effects of site-directed mutagenesis on the binding affinity and binding specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. The chitin-binding domain (ChBD) was genetically fused to the N-terminus of the green fluorescent protein, GFP. The polyhistidine-tagged hybrid protein was expressed in Escherichia coli under the dose-dependent regulation of the araBAD promoter and purified using metal affinity-, chitin- or ion-exchange chromatography. Residues suggested to be involved in binding from previous three-dimensional studies were mutated and their contributions to binding and substrate specificity were evaluated by depletion assays. Purified fusion proteins were incubated with chitin beads, polysaccharide-protein complexes were removed by centrifugation and the free protein concentration was measured fluorometrically. The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation. Binding affinity and specificity were alternatively studied by affinity electrophoresis under non-denaturing conditions. Non-conservative substitution of tryptophan residue (W687) with alanine abolished chitin-binding affinity. Double mutation E668K/P689A also impaired binding significantly. Other substitutions in the binding site had little effect on overall affinity for chitin. Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than observed for the wild-type. Furthermore, the ChBD-GFP hybrid protein proved to be useful for specifically labeling cell walls of fungi and yeast and for the detection of fungal infections in tissue samples.
Additionally, an improved method for detecting cell lytic activity by a colorbased zymogram was developed. Proteins were separated by electrophoresis in SDS-polyacrylamide gels copolymerized with Remazol-brilliant-blue labeled whole cells of Micrococcus lysodeikticus. After electrophoresis, the enzymes were allowed to refold and lyse the blue-labeled cells embedded in the gel, producing clearing zones in an otherwise bluish gel. This improved zymogram method allows the rapid, sensitive and simultaneous determination of cell lytic specificity, relative activity and molecular weight. This assay should be useful for many research disciplines investigating the role of lysozymes and other cell wall hydrolases capable of refolding after SDS treatment.
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