mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 1
1.6 Aims of the project
The main aim of this project is to better understand the properties of the multiple chaperonins
of Mycobacterium tuberculosis. These properties include the functional and oligomeric
properties of Cpn60.1 and Cpn60.2 and chimeras between them and E. coli GroEL. A list of
the topics that are addressed in this project are as follows:
Functional analysis of Mycobacterial chaperonins in E. coli. This was tested using
protein expression and complementation assays.
Functional analysis of the cochaperonin of M. tuberculosis. Since functional
cochaperonins are a vital part of the chaperoning cycle, the ability of Cpn10 to
function in E. coli was tested along with the chaperonins using complementation
assays.
Further characterisation of the proteins of M. tuberculosis by site directed
mutagenesis and domain swaps experiments to see if the ability to oligomerise can be
restored in E. coli (native gels and AUC), and the effect this has on the functional
ability of the chaperonins (complementation assays).
Testing the ATPase activity of purified functional chaperonins compared with
GroEL, as the functional cycle of GroEL is dependent on its ATPase activity.
Characterising the ability of M. tuberculosis Cpn60.1 to function in M. smegmatis.
Since Cpn60.1 and Cpn60.2 in Mycobacteria may have evolved different functions,
and the Cpn60.1 from one Mycobacteria is closely related to another Cpn60.1 from
another Mycobacteria, the ability of Cpn60.1 from M. tuberculosis to complement for
loss of Cpn60.1 in M. smegmatis was tested.
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