Insulin-like growth factor binding proteins (IGFBPs) are important modulators of IGF action. It is becoming clear that they play an important role in processes such as differentiation, growth and development. Although the six IGFBPs show a high degree of homology, little is known of their structure, interactions or functions. IGFBP1 is produced in large amounts by uterine decidua in human pregnancy. The aim of the project was to identify its ligand binding domain and to pinpoint residues which are responsible for specific binding of IGFs. This was attempted using phage display, a relatively new technique, which couples mutagenesis to functional screening. In principle, this allows desirable mutants to be selected from large pools. The method is especially suitable for studying IGFBP1, since existing knowledge of its structure is inadequate for any strategy involving directed mutations. Mammalian and bacterial expression systems were evaluated using wild-type IGFBP1, in preparation for production of mutants selected by phage display, with a view to testing structure-function relationships of IGFBP1 in vitro and in vivo to elucidate its role in pregnancy. Wild-type IGFBP1 was displayed on fd-phage and retained its IGF-binding properties. Several schemes were devised to select for IGFBP1 molecules with altered affinities for IGFI and/or IGFII, but these proved unsatisfactory for selection of IGFBP1 as IGF appears to be sequestered within the binding protein. Various random mutagenesis methods were unsuccessful, probably due to the high GC content of IGFBP1 cDNA. Hence, although the combination of random mutagenesis and phage display is a powerful technique for the screening and selection of large numbers of mutants, the technical difficulties could not be resolved in the time available.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674490 |
| Date | January 1996 |
| Creators | Butler, Georgina Susan |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35189 |
Page generated in 0.0018 seconds