All organism must respond to environmental changes and the signal transduction pathways which allow them to do so on a cellular level. The signal transduction involves second messengers for all organisms to sense and respond to their external environment. Cyclic nucleotide mainly include cAMP- The universal second messenger used by different form of life – includes; bacteria, protozoa, fungi and mammals. Hydrolytic degradation catalyses ATP by adenylylcyclases(ACs), and phosphodiesterases (PDEs) to generate cAMP.
cAMP binding proteins and their signalling is proven by in-silico methods ie, by bioinformatics studies till date. These statement of signalling mechanism of cAMP binding proteins in pathogenic organism – Vibrio cholerae , can be proven in the laboratory (wet lab) conditions by using the cyclic-AMP agarose beads showing affinity on cAMP binding proteins.
1. Harvesting the pathogenic Vibrio strain to collect intracellular components and isolation of cAMP binding intracellular protein by affinity chromatography.
2. Identified by SDS- Polyacrylamide gel electrophoresis and further characterisation using Mass spectrometry.
The proteins suspected to have bound with cAMP can be isolated and studied for its various functions and their role in signalling pathway.
• Culturing of Vibrio cholerae and lyse the cells to extract the cytosolic proteins
• Interaction of the proteins with cAMP Agarose beads
• Elution of the cAMP bound proteins using cAMP
• Protein separation using SDS-PAGE (10%) and Silver staining followed by In gel digestion
• Mass spectrometry for Identification and characterization of cAMP binding proteins
• The cAMP binding proteins can be identified by SDS-PAGE and characterize by Mass spectrometry.
• Complete biological like Biochemical analysis and Structural characterisation of cAMP binding proteins
• Hypothesis the role of those proteins for the survival of Vibrio cholera.