Protein Engineering, Vol. 12, No. 11, 981-987,
November 1999
© 1999 Oxford University Press
Elastase substrate specificity tailored through substrate-assisted catalysis and phage display
Department of Molecular Oncology, Genentech, Inc., 1 DNA Way,South San Francisco, CA 94080, USA
The catalytic histidine of human neutrophil elastase was replaced with alanine (H57A) to determine if a substrate histidine could substitute for the missing catalytic group—`substrate-assisted catalysis'. H57A and wild-type elastase were recovered directly from Pichia pastoris following expression from a synthetic gene lacking the elastase pro sequence, thereby obviating the need for zymogen activation. Potential histidine-containing substrates for H57A elastase were identified from a phage library of randomized sequences. One such sequence, REHVVY, was cleaved by H57A elastase with a catalytic efficiency, kcat/KM, of 2800 s–1 M–1, that is within 160-fold of wild-type elastase. In contrast, wild-type but not H57A elastase cleaved the related non-histidine containing sequence, REAVVY. Ten different histidine-containing linkers were cleaved by H57A elastase. In addition to the requirement for a P2 histidine, significant preferences were observed at other subsites including valine or threonine at P1, and methionine or arginine at P4. A designed sequence, MEHVVY, containing the preferred residues identified at each subsite proved to be a more favorable substrate than any of the phage-derived sequences. Extension of substrate-assisted catalysis to elastase suggests that this engineering strategy may be widely applicable to other serine proteases thereby creating a family of highly specific histidine-dependant proteases.
Keywords: human neutrophil elastase/phage display/protease specificity/substrate-assisted catalysis
1 Present address: ETH Hönggerberg, Zürich, CH-8093, Switzerland
2 To whom correspondence should be addressed; email: pjc{at}gene.com
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