Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids.
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.
Ceci
Luigi R
LR
Istituto di Biomembrane e Bioenergetica, Sezione di Trani, Via Corato 17, 70059 Trani, Italy. luigi.ceci@area.ba.cnr.it
Volpicella
Mariateresa
M
Rahbé
Yvan
Y
Gallerani
Raffaele
R
Beekwilder
Jules
J
Jongsma
Maarten A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000187:drug effects*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000235:genetics* / Q000378:metabolism / Q000469:parasitology; D010940:Plant Proteins / Q000235:genetics* / Q000633:toxicity; D013045:Species Specificity; D014357:Trypsin / Q000187:drug effects / Q000378:metabolism
C405940:MTI2 protein, Sinapis alba; D010940:Plant Proteins; D002918:Chymotrypsin; D014357:Trypsin