Substrate turnover and inhibitor binding as selection parameters in directed evolution of blood coagulation factor Xa.
A library of blood coagulation factor Xa (FXa)-trypsin hybrid proteases was generated and displayed on phage for selection of derivatives with the domain "architecture" of trypsin and the specificity of FXa. Selection based on binding to soybean trypsin inhibitor only provided enzymatically inactive derivatives, due to a specific mutation of serine 195 of the catalytic triad to a glycine, revealing a significant selection pressure for proteolytic inactive derivatives. By including a FXa peptide substrate in the selection mixture, the majority of the clones had retained serine at position 195 and were enzymatically active after selection. Further, with the inclusion of bovine pancreatic trypsin inhibitor, in addition to the peptide substrate, the selected clones also retained FXa specificity after selection. This demonstrates that affinity selection combined with appropriate deselection provides a simple strategy for selection of enzyme derivatives that catalyse a specific reaction.
Lorentsen
Rikke H
RH
Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.
Møller
Charlotte H
CH
Etzerodt
Michael
M
Thøgersen
Hans C
HC
Holtet
Thor L
TL
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D032621:DNA Shuffling; D019020:Directed Molecular Evolution / Q000379:methods*; D015951:Factor Xa / Q000235:genetics* / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013025:Glycine max; D013379:Substrate Specificity; D014357:Trypsin / Q000235:genetics; D014361:Trypsin Inhibitors / Q000378:metabolism*
D065427:Factor Xa Inhibitors; D019151:Peptide Library; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases; D014357:Trypsin; D015951:Factor Xa