Unravelling the mysteries of the milk protein β-lactoglobulin
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The over-arching aim of this thesis is to characterise and compare the biophysical and physicochemical properties of the bovine and caprine orthologues of β-lactoglobulin (βlg). This knowledge is vital to understand the different responses of cow and goat milk to digestion and processing, and differences in the immunogenicity of these milks.
Caprine and bovine βlg were successfully recombinantly expressed using specialised E. coli Origami cells conducive to the formation of disulfide bonds. Co-expression with a disulfide bond isomerase encourages the correct disulfide bonds to form. Correctly folded βlg exhibits marked stability at low pH values, thus misfolded βlg and contaminating proteins can be effectively removed by reducing the pH to below 3 and adding moderate concentrations of salt. Circular dichroism spectra and mass spectrometry confirm that each of the βlg proteins are expressed as full-length, folded constructs.
βLg contributes to milk protein allergies in humans, however, goat milk carries a lesser allergenic burden than cow milk. This may be due to differences in the structures of bovine and caprine βlg, or due to differences in the extent of digestion of these proteins. Elucidation of an ultra-high resolution crystal structure of caprine βlg reveals that it shares a very similar structure with that of the bovine variants of βlg. This suggests that antibodies raised against bovine βlg would be capable of crossreacting with caprine βlg.
Analytical ultracentrifugation studies of the solution structure of caprine βlg indicate that this protein displays a difference in self-association behaviour than bovine βlg at low pH values. Caprine βlg participates in a monomer-dimer self-association at pH values from 3.5 to 7.5, while at pH 2.5 (and 150 mM NaCl) this protein is predominantly monomeric. Bovine βlg, on the other hand, is in a monomerdimer self-association under these conditions at pH 2.5. This has the potential to affect how these proteins are presented to the immune system during digestion.
Bovine and caprine βlg display a similar level of resistance towards denaturation by urea, as assessed by circular dichroism, while molecular dynamics simulations suggest that the conformational fluctuations of these proteins occur to a similar extent and are localised to the same regions. These findings suggest that these proteins will be recognised by the immune system and acted on by digestive enzymes in the same way.
Bovine βlg is involved in the fouling of surfaces when cow milk is heat-treated. Goat milk is increasing in popularity as an alternative to cow milk, yet far less is known about the thermal denaturation processes of caprine βlg. An analysis of the thermal denaturation behaviour of caprine βlg, utilising circular dichroism spectroscopy, indicates a dependence of the thermal stability on pH that differs from that of bovine βlg. This is likely a result of the lesser net negative charge of caprine βlg compared to bovine βlg.
Despite being intensively studied for over 50 years, the physiological role of βlg in milk is still a mystery. Sedimentation velocity analyses of fluorescently-labelled βlg proteins provide evidence for a proteinprotein interaction between βlg and a component in milk. This is the first time that interactions involving βlg within milk have been investigated, made possible by the use of fluorescence-detection analytical ultracentrifugation. The co-elution of immunoglobulin proteins with βlg from milk following size-exclusion chromatography supports the hypothesis presented that βlg is capable of interacting with immunoglobulins in milk. The physiological role of βlg in milk may be to bind and protect immunoglobulins from digestion as they traverse the digestive tract. This would leave them available for absorption to support the developing immune system of new-born ruminants.