Final Activity Report Summary - SPLOCAB (Site-specific labelling of proteins for Fluorescent Tagging or Immobilisation using 'Click'-Chemistry or Staudinger Ligation)
To understand protein-protein interactions, movements and complex protein assembly in living cells is a challenge in the post-genomic era. Protein rarely functions in isolation, so protein-protein interactions affect whole processes in a cell. Many human diseases are the result of abnormal protein-protein interactions involving endogenous proteins or proteins from pathogens or both. To harness these associations is of great pharmaceutical significance. This requires detailed knowledge of each system at a molecular level. However the nature of protein interactions is complicated. Labelling the protein of interest is a good way to address this problem. In this project we are aiming to produce a new genetic chemical tool that can label protein of interest site-specifically and efficiently.
Using the strategy of bio-orthogonal chemistry, we designed and synthesized three biotin analogues bearing azide group (the chemical tools). Then these analogues have been tested as substrates for biotin ligase (BirA), an enzyme that biotinylates specific lysine residue of a 15 amino acid peptide. One biotin analogue shows excellent ligation efficiency. We have successfully introduced the 15 amino acid peptide to the protein of interest. The test of this analogue to the protein of interest is under way. Biotin is widely used in biological lab for protein purification, based on its strong non-covalent interaction with avidin.
One problem with this technique is the condition is very harsh when eluting the protein of interest from avidin column. So it is necessary to find a substitute for biotin, which has weaker binding affinity to avidin while maintains good binding affinity. Using isothermal titration calorimetry technique binding affinity of biotin analogues to avidin has been tested. Interesting results have been obtained. Using bio-orthogonal 'click' chemistry we have successfully labeled the avidin-biotin analogue complex. This biotin analogue allows us the possibility to label the protein of interest in the cell, in the meanwhile the protein can be immobilised.
Using the strategy of bio-orthogonal chemistry, we designed and synthesized three biotin analogues bearing azide group (the chemical tools). Then these analogues have been tested as substrates for biotin ligase (BirA), an enzyme that biotinylates specific lysine residue of a 15 amino acid peptide. One biotin analogue shows excellent ligation efficiency. We have successfully introduced the 15 amino acid peptide to the protein of interest. The test of this analogue to the protein of interest is under way. Biotin is widely used in biological lab for protein purification, based on its strong non-covalent interaction with avidin.
One problem with this technique is the condition is very harsh when eluting the protein of interest from avidin column. So it is necessary to find a substitute for biotin, which has weaker binding affinity to avidin while maintains good binding affinity. Using isothermal titration calorimetry technique binding affinity of biotin analogues to avidin has been tested. Interesting results have been obtained. Using bio-orthogonal 'click' chemistry we have successfully labeled the avidin-biotin analogue complex. This biotin analogue allows us the possibility to label the protein of interest in the cell, in the meanwhile the protein can be immobilised.