SPLOCAB · Site-specific labelling of proteins for Fluorescent Tagging or Immobilisation using 'Click'-Chemistry or Staudinger Ligation
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2007-01-08 → 2009-01-07
- EU contribution
- €169,365
- Participants
- 1
- Scheme
- IIF
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Results in brief
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.
Data: CORDIS, © European Union
Project objective
Yong-Qing Yang is a highly talented young chemist in the final year of her PhD at Shanghai Institute of Organic Chemistry (SIOC), Chinese Acad. Sciences, a world-leading Science & Technology Institution.She has already demonstrated significant potential to be a leading researcher through the important work that she has published in leading International chemistry journals. Her expertise in synthesis provides her with a strong platform of practical skills to complement those in chemical biology of Dr Thomas- group at Nottingham Univ. in the proposed multidisciplinary research.This project aims to develop tools to selectively modify ONLY a single protein in a cell with a functionalised biotin, and then to use this versatile biotin 'handle' to conduct further 'bio-orthogonal' chemistry (Staudinger ligation, 'click-chem') allowing the introduction of fluorescent, crosslinking or immobilisation tags ONLY onto the biotin analog. Using these tags transient protein-protein interactions and protein location in-vivo can be identified.This will provide new fundamental proteomics information forming the basis for drug discovery as fits several FP6 objectives including 'Application of knowledge and technologies in the field of genomics and biotechnology for health: rational and accelerated development of new, safer, more effective drugs'. Training at Nottingham will involve Ms Yang obtaining new practical skills in biophysical and biochemical techniques.She will then be in a strong position to act as a leader and female role model in interdisciplinary 'Chemical Biology' research in China. She will participate in the Nottingham Career Advancement program that covers managerial, IP protection and leadership skills courses for young researchers.The biotin tools developed in Nottingham will be transferred back to China, and, through collaboration applied to identify the effect of traditional Chinese medicines at a molecular level using Western Medicinal Chemistry techniques.
Original text from CORDIS.
Participants
- UNIVERSITY OF NOTTINGHAM · NOTTINGHAMCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union