Isotopically labeled bovine beta-lactoglobulin for NMR studies expressed in Pichia pastoris

Helen Denton, Mark Smith, Holger Husi, Dusan Uhrin, Paul N. Barlow, Carl A. Batt, Lindsay Sawyer

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)97-103
Number of pages7
JournalProtein Expression and Purification
Volume14
Issue number1
DOIs
Publication statusPublished - 1 Oct 1998

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