THERMOSTABLE VARIANTS OF BOVINE BETA-LACTOGLOBULIN

The thermal stability of bovine beta-lactoglobulin (BLG) has been enha nced by the introduction of an additional disulfide bond. Wild-type BL G has two disulfide bonds, C106 - C119 and C66 - C160, with a free cys teine at position 121. We have designed, with the aid of molecular mod eling 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. T hese mutants were then created by site-directed mutagenesis and purifi ed from Escherichia coli carrying a tac expression vector using a two- step renaturation method. Formation of disulfide linkages in the corre ct arrangement, as designed, was confirmed by peptide mapping. In cont rast to wild-type rBLG, which polymerizes at temperatures >65 degrees C, neither of the mutant proteins polymerized. The conformational stab ility of the L104C and A132C mutant proteins against thermal denaturat ion has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stab ility against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.