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Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids.

Abstract : The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 x 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 microg ml-1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.
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Submitted on : Monday, July 6, 2009 - 8:56:50 PM
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Luigi R Ceci, Mariateresa Volpicella, Yvan Rahbé, Raffaele Gallerani, Jules Beekwilder, et al.. Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids.. Plant Journal, Wiley, 2003, 33 (3), pp.557-66. ⟨10.1046/j.1365-313X.2003.01645.x⟩. ⟨hal-00402187⟩



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