Baker, Douglas John (1999) Chaperonins for the assisted refolding of protein. University of Southampton, Doctoral Thesis.
Abstract
The work in this thesis describes studies carried out on GroEL to produce a matrix for the refolding of proteins.
Recombinant GroEL was purified from E. coli and successfully immobilised onto insoluble matrices based upon agarose and controlled pore glass (CPG). The coupling efficiency to CPG was shown to be higher than that to agarose, CPG immobilised GroEL retained 70% of the ATPase activity of unbound GroEL and was stable for periods in excess of 6 months at 4oC. The immobilised GroEL alone was shown to arrest the refolding of chemically denatured lactate dehydrogenase (LDH), addition of ATP resulted in productive refolding, with an increased yield of LDH activity compared to LDH-refolding in free solution.
The apical domain of the GroEL homologue from Thermus aquaticus was isolated from Thermus aquaticus genomic DNA by PCR methods. The isolated gene fragment was successfully cloned into a high expression vector and expressed in E. coli. A rapid purification protocol was developed. The isolated domain unfortunately showed no refolding activity with the substrates tested, however, a protective activity was seen in shear-induced inactivation (rotary mixing). The protein was successfully crystallised in two crystal forms to diffraction quality. From a single data set the structure of the domain was determined to 2.5A resolution by X-ray crystallography and its structure is presented and compared with the same domain from GroEL.
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