Production and purification of recombinant amelogenins for investigating proteopathic mechanisms associated with amelogenesis imperfecta

In a mouse model of amelogenesis imperfecta (Al), an amelogenin p.Y64H mutation was reported to cause the abnormal retention of amelogenin in the ameloblast secretory pathway. This was hypothesised to be due to enhanced pathological aggregation of mutant amelogenin. The aim of this thesis was to develop purification methodologies to deliver large amounts of wild-type (WT) and mutant recombinant amelogenins (r-amelogenins) and develop microplate based binding assays to study protein-protein interactions of these recombinants to elucidate the effect of the mutation on aggregation. His-tagged r-amelogenin was extracted from Escherichia coli (E. Coli) with 3% acetic acid. This extract was subject to nickel affinity chromatography (targeting the His-tag); the 'gold standard' technique for purifying recombinant proteins from bacterial contaminants. The His-tag was then removed but cleavage was only ~50% efficient. Cleaved r-amelogenin unexpectedly still bound the nickel column (presumably due to the presence of di and tri histidine motifs in the amelogenin sequence) which precluded its isolation from uncleaved contaminant. Size exclusion chromatography was also trialled and also found to be ineffective. Finally, preparative SOS PAGE was found to produce cleaved r-amelogenin at single band purity on analytical SOS PAGE. After optimising the purification regime, simple and cost-effective microplate binding assays were developed initially using amelogenin rich porcine enamel matrix derivative (EMD) as a surrogate. Initially the aim was to immobilise EMD on the plate and then measure the binding of FITC-labelled EMD by simple end point fluorescence measurements. An alternative method trialled UV spectrophotometry to monitor the loss of EMD from free solution in real time as it bound EMD immobilised to the well surfaces. This latter method provided an adaptable, simple and cost-effective means of monitoring amelogenin binding and aggregation. It provided pilot data suggesting that p.Y64H mutant r-amelogenin was clearly more
aggregative than WT r-amelogenin.