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Chapter 4
4.1.4 Chimera GBI
This chimera also consists of an imprecise domain swap between Cpn60.2 and GroEL. In this
chimera however, the equatorial domain sequence and a partial segment of the intermediate
domain sequence are from GroEL, while the apical domain sequence and the remainder of
the intermediate domain sequence comes from Cpn60.2.
As with AHC, it was expected that this chimera would function in E. coli MGM100.
However, even this chimera was unable to rescue cell growth in glucose (fig 4.6a). Another
strange result was that, on SDS-PAGE gels the protein band showed some over expression,
but it always showed a band with a lower molecular weight (fig 4.6b). Sequence analysis
however, showed that there were no errors in the genetic sequence. The reason for this
unclear, but it is possible that these bands were proteolytic fragments. Also, since the
chimera’s equatorial domain is from GroEL, it was expected that the native gel would show a
band at around 840kDa, representing a double ring structure. What was observed however
was that, along with the band at around 800kDa, there were also bands present closer to
400kDa (single ring) and a very faint band around 60-70kDa (monomer) (fig 4.7). However,
since the chimera had partial sequences of the intermediate domain, it couldn’t be claimed
that it was entirely due to the apical domain that the oligomeric stability of the chaperonin
was being affected.
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