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

Chapter 3
3.3.1 Using pET plasmid in Tab21
To try and obtain a higher level of protein expression for Cpn60.1, its gene was cloned into a
pET vector to be used with Tab21. It was expected that this would allow the required gene
products to be expressed in greater quantity as compared with the pTrc plasmid by making
use of the T7 transcription and translation mechanism.
A pET vector with the ampicillin resistance gene was chosen, as Tab21 is kanamycin
resistant. It was decided that pET23a+ would be used as the cloning vector as it had the
required ampicillin resistance gene, and also had the appropriate restriction sites to allow a
direct swap of genes from the pTrc plasmids. A schematic representation of the swaps is
show in figure 3.7. As the level of protein expression of Cpn60.2 was high with the pTrc
plasmid, it was decided that only the Cpn60.1 gene along with both the cochaperonins would
be cloned into the pET vector. Once the required pET plasmid combinations were obtained
and confirmed by sequencing, they were transformed into Tab21 as per the protocol
described in section 2.4.16 and their expression levels were analysed.
Unfortunately expression of Cpn60.1 could not be distinguished on the gel (fig 3.8). Only the
over-expression of GroEL was seen from the cells containing pET-GroES-GroEL. It was also
observed that the strains carrying the pET-GroES-Cpn60.1 and pET-Cpn10-Cpn60.1
plasmids showed slower growth in both liquid and solid media compared with cells carrying
the pTrc versions. An accurate complementation analysis could not be done as cell growth
was extremely slow on solid media with glucose and arabinose. The reason for this
observation is unclear. It is possible that the loss of the cells ability to regulate its endogenous
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