Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.
We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.
Roberts
B L
BL
Protein Engineering Corporation, Cambridge, MA 02138.
Markland
W
W
Siranosian
K
K
Saxena
M J
MJ
Guterman
S K
SK
Ladner
R C
RC
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000235:genetics* / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004279:DNA, Viral; D006863:Hydrogen-Ion Concentration; D019272:Leukocyte Elastase; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D004279:DNA, Viral; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D007611:Aprotinin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D014357:Trypsin