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

Chapter 4
SDS-PAGE gels to check expression.
As was the case with the original imprecise domain swaps, it was hypothesised that the apical
domain is responsible for substrate recognition, and that the introduction of the apical domain
from Cpn60.1 into GroEL would result in this chimera failing to complement for the loss of
endogenous GroEL as it is unable to recognise the same substrates as GroEL.
The results obtained from the complementation plates of MQO were as expected. In the
presence of arabinose all the cultures grew, but in the presence of glucose when endogenous
GroEL expression is suppressed, MQO was unable to complement (fig 4.8a). The expression
levels of MQO were examined to ensure that the lack of complementation was not due to a
lack in expression. It was observed, on the SDS-PAGE gel, that the expression level of MQO
was low but still present (fig 4.8b), and this was further confirmed with MS. Although its
expression level was low, it is known that very low numbers of functional chaperonins are
needed for normal growth (Lorimer, 1996). On the native gel, a band of approximately
400kDa was observed, which is normally associated with a chaperonin that assembles as a
single ring (fig 4.9). This result suggests that the apical domain may also play some role in
the oligomeric stability of the chaperonins, as was observed with other chimeric constructs
(section 4.1.3 and 4.1.4). However, further experiments would be needed to confirm this
observation.
From the results obtained, it was reasoned that if the lack of function was in fact due to the
lack of ability to recognise appropriate substrates, then a reciprocal swap between GroEL and
Cpn60.1 should yield a fully functional chimeric chaperonin. For this purpose, another
chimera was constructed where the apical domain was from GroEL, while the equatorial and
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