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Tesi etd-10292014-181502

Thesis type
Tesi di laurea magistrale
In-vitro characterization of promiscuous biotinylation by biotin ligase
Corso di studi
relatore Bernardi, Rodolfo
relatore Mutwil, Marek
Parole chiave
  • purification
  • BirA
  • protein-protein interactions
  • biotinylation
Data inizio appello
Riassunto analitico
Protein-protein interaction networks represent useful system to better understand the organisation of the proteome and, moreover, provide a support for identifying protein functions. Thus, identification of these interactions would help to reach an improved comprehension of the entire cell. Most popular methods for detecting protein interactions are either in vitro assays or are based on heterologous systems, both of which do not represent true cellular environment. Thus, a lot of research is directed towards development of new methods that detect interactions under in vivo conditions, so as to preserve the spatio-temporal integrity of these interactions. One of such methods involve the biotin protein ligase (BirA) from Escherichia coli, which is responsible for catalysing transfer of a small molecule, biotin, to a specific lysine residue (within a specific conserved sequence) of acceptor proteins. This reaction is highly specific, with only one protein in E.coli capable of being biotinylated in vivo. A mutation in BirA (R118G; BirA*), however, reduces this specificity such that the intermediate of reaction (biotinoyl-AMP) is released from the active site of the protein and can biotinylate any protein that it encounters. Based on this principle, if we fuse a protein with BirA*, the proteins that come in proximity of the fusion construct should be biotinylated and could further be identified by a streptavidin pull-down.
In order to develop this technique in plants, the enzymatic properties of BirA* need to be characterized. The current project was focused on purifying BirA* (codon optimized for expression in Arabidopsis thaliana) from bacteria and assessing its enzymatic properties under different physiological conditions. To this end, GST-6X Histidine fused BirA* was purified using inducible bacterial overexpression system and checked for promiscuous biotinylation of non-native target, i,e., free GST protein. Further, the substrate concentration, temperature, reaction time and pH conditions for the progress of reaction were optimised. The results generated by the current study verify that BirA* can catalyse promiscuous biotinylation, therefore it would be worthwhile to extend this system in plants for detection of protein-protein interactions. Moreover, if BirA* is selectively overexpressed in a specific cellular compartment, it has potential to biotinylate most of the proteins within this compartment. As different cellular compartments differ in pH, the present study also helped to short-list the compartments whose proteome could be deciphered using BirA*.
To summarize, the present study verified that BirA* based proximity-biotinylation technique could be used to develop an in vivo assay for detecting protein interactions and to decipher organellar proteome. Also, we were able to optimize the reaction conditions at which the enzyme is most active.