Isotopically labeled bovine beta-lactoglobulin for NMR studies expressed in Pichia pastoris.
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.
Structural Biochemistry Group, The University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, United Kingdom.
H
H
Denton
M
M
Smith
H
H
Husi
D
D
Uhrin
P N
PN
Barlow
C A
CA
Batt
L
L
Sawyer
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes; D002417:Cattle; D019281:Dimerization; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010843:Pichia / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002247:Carbon Isotopes; D007782:Lactoglobulins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins