Final Report Summary - ADUKKIPA74 (In Meso Crystallogenesis of GPCR-protein ligand complexes)
A summary description of the project objectives
The current project has two main objectives which are described below. The first objective is to facilitate a transfer of technology involving recombinant human membrane protein expression in eukaryotic cells from the researcher to the host laboratory. The second objective is for the researcher to gain knowledge and skills in the field of in meso crystallization from the host laboratory.
A description of the work performed since the beginning of the project
In line with the two main objectives of the project, extensive work has been done in achieving these objectives. The researcher has set up a functional tissue culture lab that is currently being used for expression and purification of recombinant human mebrane proteins. The current expertise in this area includes maintaining eukaryotic cells such as sf9 and Hi5 insect cells in culture. At present, we can maintain long-term cultures of these cell lines for recombinant protein production purposes. The researcher also implemented laboratory protocols for the generation and amplification of recombinant baculovirus' for large-scale infection and protein production. These two aspects of the technology have been robustly tested as evidenced by the fact that we have successfully expressed and purified several human membrane proteins for structure determination purposes. The candidate has also gained theoretical and practical knowledge in the field of in meso crystallization.
A description of the main results achieved so far
The candidate has expressed and purified several human mebrane proteins. These proteins have been subjected to extensive crystallization trials. Of these, one has currently yielded in meso crystals that diffract to 4.0 A.
The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
It is expected that in the near future the protein crystals diffracting to 4.0 A will be further optimized to yield the structure of this therapeutically relevant protein. It is expected that from the structure, we can glean sufficient information to design novel small-molecule drugs to treat widely prevalent diseases such as Asthama and Cardiac Heart Failure.
The current project has two main objectives which are described below. The first objective is to facilitate a transfer of technology involving recombinant human membrane protein expression in eukaryotic cells from the researcher to the host laboratory. The second objective is for the researcher to gain knowledge and skills in the field of in meso crystallization from the host laboratory.
A description of the work performed since the beginning of the project
In line with the two main objectives of the project, extensive work has been done in achieving these objectives. The researcher has set up a functional tissue culture lab that is currently being used for expression and purification of recombinant human mebrane proteins. The current expertise in this area includes maintaining eukaryotic cells such as sf9 and Hi5 insect cells in culture. At present, we can maintain long-term cultures of these cell lines for recombinant protein production purposes. The researcher also implemented laboratory protocols for the generation and amplification of recombinant baculovirus' for large-scale infection and protein production. These two aspects of the technology have been robustly tested as evidenced by the fact that we have successfully expressed and purified several human membrane proteins for structure determination purposes. The candidate has also gained theoretical and practical knowledge in the field of in meso crystallization.
A description of the main results achieved so far
The candidate has expressed and purified several human mebrane proteins. These proteins have been subjected to extensive crystallization trials. Of these, one has currently yielded in meso crystals that diffract to 4.0 A.
The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
It is expected that in the near future the protein crystals diffracting to 4.0 A will be further optimized to yield the structure of this therapeutically relevant protein. It is expected that from the structure, we can glean sufficient information to design novel small-molecule drugs to treat widely prevalent diseases such as Asthama and Cardiac Heart Failure.