Denton H, Smith M, Husi H, Uhrin D, Barlow P N, Batt C A, Sawyer L
Structural Biochemistry Group, The University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, United Kingdom.
Protein Expr Purif. 1998 Oct;14(1):97-103. doi: 10.1006/prep.1998.0924.
beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments. At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet.
β-乳球蛋白（β-Lg）是反刍动物乳中的主要乳清蛋白，与乳蛋白的不可逆变性及其在热处理过程中相关的不良加工行为有关。为了有助于理解这种行为，并促进对分子结构与其在溶液中行为之间关系的其他研究，我们已经制备并纯化了足够量的15N标记和13C/15N双标记的β-Lg，以便使用异核核磁共振光谱法全面测定其结构和动力学。使用毕赤酵母系统对标记蛋白进行过量表达，产率良好，但需要去除大量共纯化的碳水化合物，否则会干扰核磁共振实验。在pH 2时，所得物质给出了高质量的三重共振核磁共振光谱，这与富含β-折叠的单体球状蛋白一致。
