title: Thermostable variants of bovine beta-lactoglobulin.
abstract: The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.
authors.affiliation: Department of Food Science, Cornell University, Ithaca, NY 14853.
authors.forename: Y
authors.initials: Y
authors.lastname: Cho
authors.forename: W
authors.initials: W
authors.lastname: Gu
authors.forename: S
authors.initials: S
authors.lastname: Watkins
authors.forename: S P
authors.initials: SP
authors.lastname: Lee
authors.forename: T R
authors.initials: TR
authors.lastname: Kim
authors.forename: J W
authors.initials: JW
authors.lastname: Brady
authors.forename: C A
authors.initials: CA
authors.lastname: Batt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003198:Computer Simulation; D003553:Cystine / Q000737:chemistry; D006358:Hot Temperature; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D007782:Lactoglobulins; D011993:Recombinant Fusion Proteins; D003553:Cystine