Antimicrobial proteins

Details Antimicrobial proteins Antimicrobial proteins

1996-06-12

1998-05-12

Lebuyader, John

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

530324, A61K 3800, C12N 1300

Patent

active

057505047

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 of International application PCT/GB94/02766, filed Dec. 19, 1994.
This invention relates to antimicrobial proteins, processes for their manufacture and use, and DNA sequences encoding them. In particular it relates to antimicrobial proteins capable of being isolated from seeds of Heuchera or Aesculus.
In this context, antimicrobial proteins are defined as proteins possessing at least one of the following activities: antifungal activity (which may include anti-yeast activity) or antibacterial activity. Activity includes a range of antagonistic effects such as partial inhibition or death. Such proteins may be oligomeric or may be single peptide subunits.
The genus Heuchera is part of the Saxifragaceae plant family. The Saxifragaceae is a large widespread family comprising mainly perennial herbs and shrubs, containing the currants and gooseberries as well as many popular garden flowers.
The genus Aesculus is part of the Hippocastanaceae plant family. The Hippocastanaceae is a small family of trees comprising two genera. The genus Aesculus is best known for its ornamental trees, notably the horse chestnut (Aesculus hippocastanum) whose brown seeds are the "conkers" much prized by children.
Plants produce a wide array of antifungal compounds to combat potential invaders and over the last ten years it has become clear that proteins with antifungal activity form an important part of these defenses. Several classes of such proteins have been described including thionins, beta-1,3-glucanases, ribosome-inactivating proteins, zeamatins, chitin-binding lectins and chitinases. These proteins have gained considerable attention as they could potentially be used as biocontrol agents.
International Patent Application Number PCT/GB92/01570 (published on 18 Mar., 1993 under the publication number WO93/05153, the disclosure of which is incorporated herein by reference) describes a protein class comprising antifungal proteins (AFPs) and antimicrobial proteins (AMPs). The class includes the following proteins: Rs-AFP1 and Rs-AFP2 capable of isolation from Raphanus sativus (Terras FRG et al, 1992, J Biol Chem, 267:15301-13309), BN-AFP1 and Bn-AFP2 capable of isolation from Brassica napus, Br-AFP1 and Br-AFP2 capable of isolation from Brassica rapa, Sa-AFP1 and Sa-AFP2 capable of isolation from Sinapis alba, At-AFP1 capable of isolation from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2 capable of isolation from Dahlia merckii, Cb-AMP1 and Cb-AMP2 capable of isolation from Cnicus benedictus, Lc-AFP capable of isolation from Lathyrus cicera, Ct-AMP1 and Ct-AMP2 capable of isolation from Clitoria ternatea. This protein class will hereinafter be referred to as "the Rs-AFP-type proteins". These and other plant-derived antimicrobial proteins are useful as fungicides or antibiotics, particularly for agricultural purposes. The proteins may be applied to or around a plant or may be expressed within a plant.
We have now purified two new potent antimicrobial proteins.
We have purified a new antimicrobial protein from seeds of Heuchera sanquinea, hereinafter called Hs-AFP1 (Heuchera sanquinea--antifungal protein 1). Hs-AFP1 is a 5 kDa polypeptide; such polypeptides may be associated as dimers. Hs-AFP1 shows a wide range of antifungal activity.
We have also purified a new antimicrobial protein from seeds of Aesculus hippocastanum, hereinafter called Ah-AMP1 (Aesculus hippocastanum--antimicrobial protein 1). Like Hs-AFP1, Ah-AMP1 is a 5 kDa polypeptide. Ah-AMP1 shows a wide range of antifungal activity.
According to the present invention, there is provided an antimicrobial protein having an amino acid sequence as shown in SEQ ID NO 1 or SEQ ID NO 2, or an amino acid sequence which is substantially homologous to SEQ ID NO 1 or SEQ ID NO 2 (preferably having at least 60% sequence identity) provided that such a protein has antimicrobial activity.
An antimicrobial protein according to the invention is capable of being isolated from seeds of Heuchera or Aesculus, and may also be capable of isolation from the seeds of both rel

REFERENCES:
Stitt et al., Regulation of metabolism in transgenic plants, Annu. Rev. Plant Physiol. Plant Mol. Biol., vol. 46, pp. 341-368, 1995.

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