Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

ALTERING THE SPECIFICITY OF THE MUSTARD TRYPSIN INHIBITOR MTI2 BY SITE DIRECTED MUTAGENESIS AND PHAGE DISPLAY

VOLPICELLA M.*, CECI L.R.**

* Dip. Biochimica e Biologia Molecolare, Università di Bari

** Centro di Studio sui Mitocondri e Metabolismo Energetico-CNR Sezione di Trani, Bari

Plant proteinase inhibitors (PIs) are low molecular weight proteins generally detected in storage and reproductive organs and induced in leaves after insect and pathogen attacks. In the past years this natural defense mechanism has been the base for the production of transgenic plants by plant-to-plant gene transfer (1). The major drawback of this approach relies on the insect capacity to produce new and “insensitive” proteinases when reared on transgenic plants or on artificial diets containing PIs (2). New studies are therefore necessary to understand the insect adaptation response to PIs ingestion. In particular, differences between sensitive and insensitive proteinases and their interaction with PI need to be clarified.

Even if more than ten different families of plant PIs active against serine proteinases have been identified, a gene coding for a trypsin inhibitor belonging to a new family of PIs has been detected in mustard (3). Thereafter inhibitors (or genes) highly homologous to the mustard inhibitor MTI2 have been detected in other Cruciferae (rapeseed and Arabidopsis thaliana) (4).

Aim of our work is the set up of a “phage display” based selection of MTI2 variants able to inhibit specific insect insensitive proteinases. In this technique, the mti2 gene is firstly randomized around the enzyme-binding site and inserted in phagemide vectors fused to a phage coat protein gene. New specific inhibitors can be selected after displaying the inhibitor variants on phage surfaces and selectively enriching the mixture for those phages that will strongly bind to an immobilized target insect proteinase. This approach carries a great deal of promise, because it provides access to millions of novel MTI2s in a single test tube.

Before randomization, mti2 variants were produced by site directed mutagenesis of codons thought important in the proteinase/inhibitor interaction, and expressed as recombinant proteins.

PIs, acting in a substrate-like way, are able to complex and inhibit specific proteinases. P1-P1’ indicates the potential reactive-site peptide bond hydrolysed by the proteinase. Residues around it play a role in determining the strength of the proteinase/inhibitor interaction.

In order to verify the reactive site of MTI2 and to study the relative importance of its amino acids, residues corresponding to P2-P1-P1’ positions of the MTI2 inhibitor (P¹⁹R²⁰I²¹) were changed by site directed mutagenesis obtaining MTI2-P¹⁹A²⁰I²¹, MTI2-P¹⁹L²⁰I²¹ and MTI2-A¹⁹A²⁰A²¹ mutants. Mutants were expressed in-vitro and assayed against trypsin and chymotrypsin.

The MTI2-WT and its mutant with the lowest activity (MTI2-A¹⁹A²⁰A²¹) were used to assess the optimal conditions for phage display selection. A successful pilot phage display selection experiment was carried out with a fake library obtained by mixing MTI2-WT and MTI2-A¹⁹A²⁰A²¹ displaying phages in a 1:10,000 ratio.

A MTI2 phage display library was then constructed by randomizing codons A¹⁸P¹⁹R²⁰I²¹F²². A library of 9.3x10⁷ independent colonies was obtained. Results of a selection of MTI2 variants by screening against immobilized trypsin and chymotrypsin will be presented.

1. Jouanin L et al. (1998) Plant Science,131, 1-11.

2. Jongsma M and Boulter C (1997) J. Insect Physiol., 43, 885-895.

3. Ceci LR et al. (1995) FEBS Letters, 364, 179-181.

4. Ceci LR et al. (1999) Proceedings of 10th International Rapeseed Congress. Canberra – Australia.

Acknowledgements-This work was funded by the EU project FAIR6-CT98-4239