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Novel pest control based on insect immune suppression and endocrine disruption - LK0948

The applicants have established an entirely novel fusion protein technique that allows biologically active peptides and proteins, which have low, or no, toxicity when delivered orally to insects, to be converted into effective and specific orally active insecticides. Thus, the purpose of this LINK project is to initiate full commercial development and to stimulate practical uptake of fusion protein technology for the benefit of UK and European agriculture. We propose, in collaboration with Isagro-Ricerca, to conduct research into the discovery, production, purification and evaluation of novel, insecticidal fusion proteins, that are effective against a wide range of UK and European pest insects, and which are less hazardous to the environment and to non-target species than current broad-spectrum insecticides. We further propose, again in collaboration with Isagro, to develop large-scale production and purification technologies, in order that further testing of fusion proteins against a wider range of UK and European pests can be accomplished, and that associated studies such as stability testing, the development of commercial formulations and or alternative delivery systems, and preliminary environmental impact assessments, etc., can be made.
Objective 1. To develop large-scale production and purification technologies for fusion proteins.
Based upon some preliminary “pilot scale” experiments (which are currently the subject of a commercial contract between Isagro and CSL/Durham, Isagro Ricerca, conduct research with the aim to develop an industrial method for a large-scale production of FP1 (1.1) and develop industrial scale purification technologies (1.2). Consequently, a competitive cost will be achieved for the resulting fusion protein product. Hence selected strains of Pichia pastoris, obtained from CSL and Durham, will be fermented in order to reach the highest content of biomass enrichment in the FP (Isagro). In this way, Isagro will employ its experience of optimising traditional parameters (e.g. composition of nutrients, gas content, etc.) in order to develop a practical method for large-scale production of fusion proteins.
The subsequent purification techniques will be oriented by appropriate studies, following the determination of a minimum level of purity required to obtain a reliable insecticidal activity (1.3) so as to minimise the production costs. During all the stages of this research some inert or other components could be added to make easier the manufacturing steps as well as to get a product with a reasonable shelf-life duration, which can assure a successful commercialisation (see also objective 8).

Objective 2. To identify and isolate novel, naturally occurring, proteins and peptides with insecticidal activity against representative pests of arable crops.
Previous research in the Defra (PSD)-funded programme PI03, and more recently in the project PS2102, has provided several lead peptides with potential insecticidal, development-disrupting, or immunosuppressive activities. For example, saliva of the predatory bug Podisus maculiventris contains a highly active insecticidal molecule with rapid paralysing activity, and the venom gland of the parasitoid Eulophus pennicornis contains a potent anti-moulting factor. Similarly, work at Durham University has identified other molecules with potential for incorporation into insecticidal fusion proteins. In the first phase of this objective (2.1) we will select 2 of the most promising candidate peptides and proteins for incorporation into insecticidal fusion proteins (CSL and Durham). Two sources of insecticidal molecules will be exploited. First, existing databases will be searched for encoding DNA or amino acid sequences of peptides/proteins, which would be insecticidal if delivered to the insect haemolymph. Secondly, to generate novel insecticides, toxic molecules produced by predators or parasites of target insect species will be purified from appropriate organs (e.g. venom glands, fat body, teratocytes, etc.) of the selected sources (2.2) by established techniques (CSL and Durham). Simultaneously, (2.3) bioassay techniques will be developed to enable monitoring of biologically active fractions during purification (2.4) (CSL and Durham). While complete purification of the toxic peptides/proteins is desirable, it is unlikely to be necessary, since peptides/proteins can be characterised from complex mixtures through the use of LC-MS, MALDI-TOF MS and Q-TOF MS (2.5) (CSL and Durham). N-terminal sequencing will also be used if necessary to give additional sequence information. The aim will be to obtain sufficient amino acid sequence data (2.6) from the toxic peptide/protein to allow their encoding genes to be isolated (see objective 3).

Objective 3. To isolate and clone the genes encoding insecticidal peptides and proteins.
DNA sequences encoding the selected toxic peptides/proteins will be obtained by one of three strategies. Where a sequence has already been lodged in the databases, it will be obtained directly, or amplified from a suitable source tissue by PCR, using primers based on the database sequence. In those cases where partial amino acid sequence is available, a complete encoding cDNA will be obtained by probing a cDNA library constructed from the appropriate tissue source. A nucleotide probe will be produced by PCR, using oligonucleotide primers based on the partial amino acid sequence of the peptides (3.1) (CSL and Durham). Alternatively, 5’- and 3’-RACE will be carried out to convert a partial cDNA (either produced by PCR, or by probing a library) into a full-length coding sequence. As a further alternative, where the toxin is a relatively short protein or peptide (<50 aa), and the amino acid sequence can be fully determined, a synthetic gene encoding the peptide/protein will be produced by oligonucleotide synthesis. In all cases, the full-length coding sequence will be cloned, fully sequenced, and transferred to a suitable expression vector to allow recombinant peptide/protein to be produced to confirm its biological activity (3.2) (CSL and Durham).

Objective 4. To construct novel fusion proteins incorporating genes encoding novel insecticidal peptides and proteins.
Recombinant genes encoding novel fusion proteins, combining one of the toxic peptides/proteins obtained under objective 3 with the plant lectin binding/transport protein GNA, will be constructed using the techniques already established by the applicants (4.1). The fusion proteins will be produced in functional form in appropriate expression systems (either E. coli or Pichia pastoris) and purified (4.2) (CSL and Durham). Further methodological details of these techniques are presented in Appendix A. Selected fusion proteins will also be expressed in transgenic potatoes, as an alternative system for protein production, and to develop plants with endogenous pest resistance.

Objective 5. To evaluate novel fusion proteins for biological activity against a range of UK pests of arable crops.
Selected (active) fusion proteins (or plants expressing selected fusion proteins) will be assayed against a range of UK arable crop pests using established insect bioassay techniques developed during the previous assessment of fusion protein efficacy (previous Defra projects in the former PI03 and PS2102 projects) (CSL and Durham), and/or by established screening methods developed by Isagro for the commercial evaluation of new insecticidal products (Isagro). The precise selection of appropriate target species will to some extent depend on the nature and mode of action of each new fusion protein, however, we aim to test promising candidate fusion proteins against at least one UK pest species from the Orders Lepidoptera (e.g. Mamestra brassicae, Agrotis segetum or Plutella xylostella), Coleoptera (e.g Psylloides chrysocephala and Otiorynchus sulcatus), Diptera (e.g. Delia radicum) and Hemiptera/Homoptera (e.g. Myzus persicae) (CSL and Durham). (5.1) Additional evaluations will be made by Isagro against selected European pests (e.g. Leptinotarsa decemlineata) (Isagro) (5.2).

Objective 6. To devise chemical routes for site directed linking of fusion protein components.
Chemical methods for stable site-directed linking of fusion protein components based on self-splicing intein proteins will be used to link potentially toxic peptides produced by an automated amino acid synthesiser to a suitably modified carrier lectin binding/transport protein produced in a recombinant protein expression system (6.1) (Durham). Initial experiments will use a bacterial lectin produced in E. coli, but the system will be extended to allow functional plant lectins suitable for linking chemically to peptides to be produced in the yeast Pichia pastoris (6.2) (Durham). This system will allow peptides with modified amino acids, such as amidated C-termini, to be incorporated into fusion proteins; recombinant protein expression systems do not allow modified amino acids to be easily incorporated. In addition, if GNA and Manse-AS are available as purified proteins, established techniques in the coupling of proteins could be applied by Isagro Ricerca to obtain alternative syntheses of the FP relevant to the present project (6.3).

Objective 7. To isolate novel natural lectins (and the genes encoding them) from selected plant species with a view to their incorporation in fusion proteins.
Because of uncertainties about the longer-term IP status of our “lead” plant carrier lectin (GNA), we propose to consider alternative lectins for incorporation in future fusion protein molecules. The uncertainties about GNA are two-fold. First, we are not clear about the proprietary “rights” to this molecule and, although these doubts do not detract from our IP rights on the basic “platform technology” covered by the patent application, it would be prudent to develop a suitable alternative at an early stage. Second, although GNA is non-toxic towards rats in feeding trials described in the scientific literature, the source material is not a normal dietary component, and potential effects of GNA on human health cannot be ruled out without further studies. We (CSL and Durham) have identified two promising plant sources of new lectins. Details of these plant species must at this stage remain confidential, but we have evidence that the plants do contain lectins, and that the tissues are edible (i.e. non-toxic). For reasons of protecting IP options (and possible future patent applications), we will refer to these species as “plant species A” and “plant species B.” Durham University has considerable experience in the isolation and characterization of plant lectins. Seeds or tissues of the plants in question will be obtained from confidential sources, and plants will be raised to an appropriate stage of development (7.1) (CSL) prior to harvesting plant tissues. Lectins will be purified from extracts of plant tissues using established chromatographic techniques (7.2) (Durham University). Lectins will be “screened” for mannose binding activity, and an abundant and suitably active lectin will identified, partially sequenced, and the gene isolated by oligonucleotide probing of plant tissue cDNA libraries (7.3) (Durham University). Genes encoding other plant lectins, which could be considered as further alternatives, are available at Durham. If appropriate, the isolated lectin gene will be incorporated into a “prototype” fusion protein incorporating a biologically active peptide or protein (7.4) (Durham and CSL).

Objective 8 To conduct preliminary stability studies on selected fusion proteins in order to provide background information for the development of appropriate formulations and application/delivery technologies.
Isagro Ricerca has a long-standing experience in Bt-based and microrganism-based formulations. As a first step a compatibility chart of the FP at an established purity will be obtained (see objective 1) in order to arrange a proper formulation (a WG formulation is preferable). Any prototypes will be checked for stability (accelerated storage stability according to CIPAC n° MT 46) (8.1), hence a preliminary study to establish an analytical method for the determination of the FP content in the formulation will be the earliest objective (8.2).
In case of failure of any traditional formulation method, alternative application/delivery technologies will be tested, for example PEGlation, inclusion, coacervation, etc.
Time-Scale and Cost
From: 2004

To: 2007

Cost: £698,187
Contractor / Funded Organisations
University - Durham, Isagro-Ricera S.r.I, Central Science Laboratory
Arable Farming              
Crop Pests