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

Chapter 3
The results from these complementation plates showed that Cpn60.2 can complement in
MGM100 under appropriate conditions. However, Cpn60.1 failed to complement,
irrespective of which cochaperonin it was co-expressed with. There are two possible reasons
for this result. Either Cpn60.1 was incapable of recognising and/or folding the required
GroEL substrates or the expression level of Cpn60.1 was too low. Therefore, the levels of
Cpn60.1 expression were estimated from SDS-PAGE gels of whole cell lysates. It was
expected that the expression of all the chaperonins and cochaperonins from the pTrc plasmid
would increase in the presence of IPTG, and that this would be distinguishable on a SDS-
PAGE gel. A lower concentration of IPTG (0.1 mM) was used to reduce cell death in the
over-night cultures. The results obtained from the SDS-PAGE gels showed that the
expression of Cpn60.2 was distinguishable with both cochaperonins. The expression level of
Cpn60.1 however, was much lower and could only be seen when co-expressed with GroES
(fig 3.4a). MS confirmed that the band was Cpn60.1. As a result, it could be speculated that
the level of expression of Cpn60.1 may not have been high enough to sustain cell growth.
However a point to note here is that although the level of expression of Cpn60.1 was low, it
was not nonexistent. Interestingly, it was also observed that MetE was over-expressed in
cells expressing Cpn60.1 both with and without IPTG, but only in cells expressing Cpn60.2
when IPTG was not present (fig 3.4b). When Cpn60.2 is over-expressed with IPTG, the MetE
band cannot be seen. This further supports the hypothesis that Cpn60.2 can function as a
chaperonin in E. coli while Cpn60.1 cannot.
Based on the results obtained in this experiment it can be concluded that Cpn60.2 from M.
tuberculosis is capable of functioning as a chaperonin in E. coli. Cpn60.1 was unable to
complement for the loss of endogenous GroEL, but this could be attributed to its low
expression levels. To try and remedy this lack of expression various experiments were
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