ISOTOPICALLY LABELED BOVINE BETA-LACTOGLOBULIN FOR NMR-STUDIES EXPRESSED IN PICHIA-PASTORIS

beta-lactoglobulin (beta-Lg) is the major whey protein in ruminant mil k and has been implicated in the irreversible denaturation of milk pro teins and its associated poor processing behavior during heat treatmen t. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure a nd its behavior in solution, we have prepared and purified N-15-labele d and C-13/N-15-double-labelled beta-Lg in sufficient quantities to pe rmit a full determination of the structure and dynamics using heteronu clear NMR spectroscopy. The overexpression of the labeled protein usin g the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which o therwise interfere with the NMR experiments. At pH 2, the resulting ma terial gives triple resonance NMR spectra of good quality that are con sistent with a monomeric, globular protein rich in beta-sheet. (C) 199 8 Academic Press.