Testing the structural basis of lysozyme recognition by antibody HyHEL-5

The binding of murine monoclonal antibody HyHEL-5 to various lysozymes has been the subject of extensive study. Our laboratory has used isothermal titration calorimetry (ITC) and fluorescence spectroscopy to characterize the thermodynamics and kinetics of the antibody/antigen interaction. The x-ray crystal structure of the HyHEL-5 Fab/HEL complex (Sheriff, et al. (1987) PNAS 80, 8075-8079) shows salt links between a Glu residue at position 50 of the heavy chain and two Arg residues of HEL at positions 45 and 68. [n addition, Glu35 of the heavy chain of the Fab is thought to play a major role. In order to understand the role of these contacts in the interaction, the HyHEL-5 Fab genes have been cloned and the proteins overexpressed in E. coli using a T7-based vector system. Each chain is expressed separately and the whole Fab is formed by denaturation and co-refolding, followed by purification on a metalchelating column. The thermodynamics of association of the rFab and hen egg [ysozyme or bobwhite quail lysozyme have been characterized through 1TC. In both cases, the enthalpy of association is negative and declines linearly with temperature. Proteins bearing neutralizing mutations at heavy chain positions 50 and 35 have been obtained by PCR-based mutagenesis and are presently being characterized.