This study had two aspects. The first was the production and purification of TIMP-l. The second was a series of biophysical studies of TIMP-l and a TIMP-l derived peptide. A monoclonal antibody affinity column was developed and used to purifY large quantities of human TIMP-l for further experiments. Two E.coli expression systems were studied to determine whether they would be suitable for large scale production of recombinant protein. In the first system TIMP-I was to be secreted as a fusion protein which could be cleaved, leaving a free N-terminus. It was discovered that it was not possible to cleave off the fusion protein. In the second system, the protein was secreted, without additions to the periplasm. Although active protein, with the correct N-tenninus, was obtained, the yields were too low to be of use for large scale expression. Secondary structure analysis by CD and FTIR showed TIMP-l to be a mostly f3- sheet protein (approaching 50%) with around 20% a-helix. A temperature study using these techniques found that little change occurs until temperatures of over 60°C where the protein aggregates. The small changes appear to be a general loosening of the structure. In analyses of the surface of TIMP-l, additional carbohydrate was identified (other than the two N-linked chains) using Con-A probing of Western blots. TIMP-l purified from WI-38 foetal lung fibroblast cells can be separated into two pools by Concanavalin A-Sepharose chromatography. These two pools were found to have a different set of pIs and a different monosaccharide composition. The use of NMR paramagnetic probes identified a hydrophobic region exposed on the surface of TIMP-I. This region probably includes a tyrosine residue and either a tryptophan or phenylalanine. The presence of an exposed hydrophobic region was also shown in binding studies using the fluorescent probe ANS. These studies identified a single, low affinity binding site. An additional study with the N-terminal fragment of type-I collagenase found no binding sites on the enzyme, but a change in fluorescence occurred when TIMP-I was present. A peptide was designed based on the N-terminal sequence of TIMP-I. High homology, susceptibility to mutation and an interesting resemblance to the Bowman-Birk family of inhibitors suggested that this peptide might be inhibitory. It was found to have only a weak inhibitory activity against gelatinase. NMR studies of this peptide in water showed a large number of conformers as a result of stabilisation of the cis isomer of its proline residues. This preference for the cis form was retained for one proline in the solvent, TFE. Preliminary NMR studies were also carried out which concluded that TIMP-I should be suitable for further structural studies using isotopic labelling.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:282777 |
| Date | January 1995 |
| Creators | Hodges, Deborah Jane |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://oro.open.ac.uk/57546/ |
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