β-lactoglobulin (BLG) is the major whey protein in ruminants and is found in the milk of a wide range of species. Extensive study of BLG over the years has led to the determination of the structure of the protein at 2.8AA and has revealed its ability to bind a variety of small hydrophobic molecules. However, the physiological function of BLG remains unknown despite its inclusion within the lipocalycin family on the grounds of genetic and tertiary structure comparisons as well as similarities in amino acid sequence. A protein engineering approach was adopted to study the residues involved in subunit interactions and ligand binding as well as the importance of Cys119 and 121 in determining the stability of the tertiary structure. The complete coding sequence for ovine BLG was obtained and a convenient site-directed mutagenesis system was set up. An expression system in the yeast <i>Saccharomyces cerevisiae</i> was developed in which ovine BLG was synthesised and secreted into the growth medium. Secretion was directed using the native BLG leader sequence. N-terminal sequencing of the secreted protein showed that the excision of the signal peptide occurred at the same site observed <i>in vivo</i>. Four mutant forms of the protein were generated. Cys119 and Cys121 were replaced with serine as separate mutants. Ile29, one of the residues at the subunit interface, was replaced with aspartate, and Lys70, located at the entrance to the hydrophobic pocket, was replaced with asparagine.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660407 |
| Date | January 1992 |
| Creators | Paterson, Gary J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/20092 |
Page generated in 0.0135 seconds