Selection of potent chymotrypsin and elastase inhibitors from M13 phage library of basic pancreatic trypsin inhibitor (BPTI).
The combinatorial approach offered by phage display has proved to be powerful in obtaining novel variants of canonical inhibitors of serine proteinases that show new binding patterns. We applied this strategy to search for variants of basic pancreatic trypsin inhibitor (BPTI) that would be strong inhibitors of two serine proteinases: bovine alpha-chymotrypsin and porcine pancreatic elastase. BPTI only moderately inhibits the first and does not inhibit the second enzyme. A representative library of 3.2 x 10(4) BPTI variants, randomized at P(1), P(1)', P(2)' and P(3)' positions of the proteinase binding loop, was displayed on the surface of phage M13. After four to five rounds of selection on the target proteinase consensus sequences of the inhibitor binding loop were obtained. In both cases, the variants selected differed from BPTI at two to four positions, with a strong preference for selection of hydrophobic residues. Nevertheless, five of these variants expressed in a free form appeared to be correctly folded, stable proteins, and did not aggregate during thermal denaturation. The midpoints of the thermal unfolding curves of these variants were lowered by 5-20 degrees C as compared to BPTI. The expressed variants proved to be new potent inhibitors of the target enzymes with association constants up to 6.9 x 10(9) M(-1) and 3.7 x 10(10) M(-1) for elastase and chymotrypsin, respectively. Thus, the inhibitory properties of BPTI were improved by as much as 7 x 10(6)-fold towards elastase and 420-fold towards chymotrypsin.
Kiczak
L
L
Lavoratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
Kasztura
M
M
Koscielska-Kasprzak
K
K
Dadlez
M
M
Otlewski
J
J
D000818:Animals; D007611:Aprotinin / Q000235:genetics*; D017104:Bacteriophage M13; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004795:Enzyme Stability; D008958:Models, Molecular; D009154:Mutation; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D019151:Peptide Library; D011485:Protein Binding; D013696:Temperature
D019151:Peptide Library; D007611:Aprotinin; D002918:Chymotrypsin; D010196:Pancreatic Elastase