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Lenger, Janina: Activity-based sulfatase and affinity-based metalloprotease probes: ready for take-off. 2011
Inhalt
Abstract
Introduction to Activity- and Affinity-Based Proteomics
Probe Design – the Crucial Step
Targeting Groups
Tagging Strategies
Probe Assembly and Linker Design
Targeting Sulfatases
Introduction to Sulfatases
Activity-Based Studies of Sulfatases
Objectives
Synthesis of Sulfatase Probes
Quinone Methide Precursors
Cyclic Sulfamate Probes
Azido Reporter Groups and in situ CuAAC
Conclusion of Sulfatase Probe Synthesis
Biochemical Evaluation of Quinone Methide Type Probes
19F-NMR Experiments
Inhibition Studies
Labeling Results
Conclusion of QM-Type Probe Experiments
Cyclic Sulfamates as Sulfatase Targeting Groups
Inhibition Experiments
Investigation of the Inhibitiory Mechanism
Preliminary Sulfatase Labeling Results
Conclusion of Cyclic Sulfamate Probe Investigations
Experimental
Synthesis of Quinone Methide Precursor Probe 1b
Synthesis of Cyclic Sulfamate Probe Components
Biochemical Evaluation of QM Precursors
Biochemical Evaluation of Cyclic Sulfamates
Expression and Affinity-Based Labeling of Arabidopsis thaliana MMPs
Introduction to Matrix Metalloproteases
Affinity-Based Studies of Matrix Metalloproteases
Objectives
Development of Covalently-Binding Marimastat Probes
Transient At-MMP Expression
Photochemical Affinity-Based Labeling of At-MMPs
Conclusion
Experimental Section
Probe Evaluation
Expression of At-MMPs
Labeling of At-MMPs with Probe 3
General Experimental Section
Synthesis: Analytical Methods, Solvents, and Reagents
Biochemical Material
General Biochemical Methods
Electrophoresis
Protein Analysis
Bacteria Cultivation
Polymerase Chain Reaction
Transformation of Bacteria
Transient Expression of Proteins in N. benthamiana by A. tumefaciens Infiltration
Conclusion