THE REGION AROUND RESIDUE-115 OF HUMAN BACTERICIDAL PERMEABILITY-INCREASING PROTEIN IS NOT INVOLVED IN LIPOPOLYSACCHARIDE-BINDING OR BACTERICIDAL ACTIVITY - CHEMICAL SYNTHESIS AND EXPRESSION OF A GENE CODING FOR THE ACTIVE DOMAIN AND CHARACTERIZATION OF RECOMBINANT PROTEINS

Bactericidal/permeability-increasing protein (BPI) is a potent antimic robial agent produced by polymorphonuclear leucocytes that specificall y interacts with and kills Gram-negative bacteria. An 825 bp gene dete rmining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli. Th e recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined. Folding wa s critically dependent on the urea and salt concentrations as well as the pH. BPI' bound with high affinity to Salmonella typhimurium cells (apparent K(d) = 36 nM), permeabilized their outer membranes to actino mycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity. In contrast with the native prote in, however, it could not be efficiently released from the cell surfac e by the addition of high concentrations of Mg2+ ions. Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxici ty. However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able t o function even when presented to cells in an unfolded form. A BPI' de rivative, containing a 13-residue foreign antigenic determinant geneti cally inserted between Ala115 and Asp116, was also produced. The deriv ative was functional in the above assays and bound with high affinity to S. typhimurium (apparent K(d) = 74 nM). These results imply that th e region defined by these residues is not involved in the lipopolysacc haride-binding or bactericidal activities of BPI. The availability of functional, non-glycosylated recombinant derivatives of BPI should gre atly aid detailed studies on its structure, interactions with lipopoly saccharide and mechanism of action.