The existing medium resolution structure of the predominant bovine milk whey protein, ß- lactoglobulin (Blg) showed that the fold of the monomer consisted of a
barrel of 9 ß- strands and one 3 -turn a -helix. Its similarity to retinol binding protein
(RBP), two bilin binding proteins and its close sequence similarity to other transport proteins, has resulted in its inclusion in a superfamily, the lipocalycins. The ß- barrel
forms an hydrophobic pocket which is thought responsible for its ability to bind a
series of small hydrophobic molecules. The function of the protein is uncertain.
The possibility of engineering the protein's hydrophobic pocket so that it can carry
small hydrophobic drug molecules through the stomach, where their presence may be
detrimental is investigated in the following manner.
Digestion experiments indicated that the protein has remarkable resistance to bovine
pepsin and, to a lesser extent, trypsin. Its resistance to human pepsins is such that it
would allow its passage through the stomach without any degradation. The presence
of a ligand bound to the protein was shown to enhance the resistance to the protease
The solution of two small molecule structures is described and serves as an
introduction to the technique of X -ray crystallography. Refinement of the existing
model, lattice Y at pH 7.8 (space group B2212, a =55.7 A, b =67.2 A, c =81.7 A)
encounters some problems and these are discussed. A new medium resolution data
set was collected and allows a more accurate model to be obtained. Refinement with
the least squares package TNT gives an R- factor of 20.1% at 3.0 A. The resealing of
existing high resolution data is described which will be merged with the medium
resolution data set.
Crystals of the protein were grown from ammonium sulphate at pH 3.0 (space group
P63; a =b =68.49 A; c= 143.17 A) and data have been collected. A low pH structure
will help investigate the proteins remarkable stability under these conditions. It will
also help in the investigation of residues within the hydrophobic pocket which may be
genetically engineered. Data have also been collected on crystals grown at the
proteins isoelectric point, pH 5.2 (space group P21; a =72.2 A; b =67.9 A; c =36.2 A;
ß= 92.0 °), and examined by molecular replacement which orientates the lattice Y
structure in the cell of the unknown.
Blg is the most antigenic milk protein and the purification of monoclonal antibodies
towards Blg was undertaken to allow future crystallisation of a complex between the
antigen binding fragment and Blg. Such a complex will reveal an antigenic site and
allow investigation of the interaction.
The final chapter discusses the results obtained and concludes that Blg is a good
candidate to engineer for the production of a drug carrier.