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
Sy. Qi et al., 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, Biochemical journal, 298, 1994, pp. 711-718
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.