Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-08-07
2003-08-12
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C435S430000, C435S069100, C435S252300, C435S252330, C435S320100, C435S410000, C435S419000, C800S294000, C800S278000, C800S280000, C800S281000, C800S290000, C536S023600, C536S024300
Reexamination Certificate
active
06605698
ABSTRACT:
This application is a 371 of PCT/GB96/03068, filed Dec. 12, 1996 and claims prior to foreign application numbers UK/9525455.3, filed Dec. 13, 1995 and UK/9606552.9, filed Mar. 28, 1996.
This invention relates to antifungal proteins, processes for their manufacture and use, and DNA sequences encoding them.
In this context, antifungal proteins are defined as proteins or peptides possessing antifungal activity. Activity includes a range of antagonistic effects such as partial inhibition or death.
A wide range of antifungal proteins with activity against plant pathogenic fungi have been isolated from certain plant species. We have previously described a class of antifungal proteins capable of isolation from radish and other plant species. These proteins are described in the following publications which are specifically incorporated herein by reference: International Patent Application Publication Number W093/05153 published Mar. 18, 1993; Terras FRG et al, 1992, J Biol Chem, 267:15301-15309; Terras et al, 1993, FEBS Lett, 316:233-240; Terras et al, 1995, Plant Cell, 7:573-588. The class includes Rs-AFP1 (antifungal protein 1), Rs-AFP2, Rs-AFP3 and Rs-AFP4 from
Raphanus sativus
and homologous proteins such as Bn-AFP1 and Bn-AFP2 from
Brassica napus
, Br-AFP1 and Br-AFP2 from
Brassica rapa
, Sa-AFP1 and Sa-AFP2 from
Sinapis alba
, At-AFP1 from
Arabidopsis thaliana
, Dm-AMP1 and Dm-AMP2 from
Dahlia merckii
, Cb-AMP1 and Cb-AMP2 from
Cnicus benedictus
, Lc-AFP from
Lathyrus cicera
, Ct-AMP1 and Ct-AMP2 from
Clitoria ternatea
. The proteins specifically inhibit a range of fungi and may be used as fungicides for agricultural or pharmaceutical or preservative purposes.
It has been proposed that this class of antifungal proteins should be named plant defensins (Terras F.R.G. et al 1995, Plant Cell, 7 573-583) and these proteins have in common a similar motif of conserved cysteines and glycines (Broekaert W. F. et al 1995, Plant Physiol. 108 1353-1358).
FIG. 1
shows the amino acid sequences of the protein Rs-AFP1 (SEQ ID NO: 34) and the substantially homologous proteins Rs-AFP2 (SEQ ID NO: 35), Rs-AFP3 (SEQ ID NO: 36), Rs-AFP4 (SEQ ID NO: 37), Br-AFP1 (SEQ ID NO: 38), Br-AFP2 (SEQ ID NO: 39), Bn-AFP1 (SEQ ID NO: 40), Bn-AFP2 (SEQ ID NO: 41), Sa-AFP1 (SEQ ID NO: 42), Sa-AFP2 (SEQ ID NO: 43) and At-AFP1 (SEQ ID NO: 44) which are small 5 kDa polypeptides that are highly basic and rich in cysteine.
FIG. 1
numbers the positions of the amino acid residues: the dash (-) at the start of the Rs-AFP3 (SEQ ID NO: 36) sequence indicates a gap introduced for maximum alignment. The sequences shown for Br-AFP1 (SEQ ID NO: 38), Br-AFP2 (SEQ ID NO: 39), Bn-AFP1 (SEQ ID NO: 40), Bn-AFP2 (SEQ ID NO: 41), Sa-AFP1 (SEQ ID NO: 42), Sa-AFP2 (SEQ ID NO: 43) and At-AFP1 (SEQ ID NO: 44) are not complete: only the N-terminal sequences are shown. The question mark (?) in the Br-AFP2 (SEQ ID NO: 39) sequence indicates a non-standard amino acid which the sequencing could not assign and which is thought to be a post-translational modification on one of the standard amino acid residues.
Further examples of antifungal plant defensins are described in International Patent Application Publication Number W095/18229 published Jul. 6, 1995 which is specifically incorporated herein by reference. These examples include Hs-AFP1, an antifungal protein capable of isolation from seeds of Heuchera species and Ah-AMP1, an antimicrobial protein capable of isolation from seeds of
Aesculus hippocastanum
. The proteins specifically inhibit a range of fungi and may be used as fungicides for agricultural or pharmaceutical or preservative purposes.
FIG. 9
shows the amino acid sequences of the proteins Hs-AFP1 and Ah-AMP1.
FIG. 9
numbers the positions of the amino acid residues. The Hs-AFP1 sequence shows 48% sequence identity with Rs-AFP1. The Ah-AMP1 sequence shows 54% sequence identity with Rs-AFP1. Hs-AFP1 shows 52% identity to Ah-AMP1 on the amino acid sequence level.
The primary structures of the two antifungal protein isoforms capable of isolation from radish seeds, Rs-AFP1 and Rs-AFP2, only differ at two positions: the glutamic acid residue (E) at position 5 in Rs-AFP1 is a glutamine residue (Q) in Rs-AFP2, and the asparagine residue (N) at position 27 in Rs-AFP1 is substituted by an arginine residue (R) in Rs-AFP2. As a result, Rs-AFP2 has a higher net positive charge (+2) at physiological pH. Although both Rs-AFPs are 94% identical at the amino acid sequence level, Rs-AFP2 is two- to thirty-fold more active than Rs-AFP1on various fungi and shows an increased salt-tolerence. The proteins Rs-AFP3 and Rs-AFP4 are found in radish leaves following localized fungal infection. The induced leaf proteins are homologous to Rs-AFP1 and Rs-AFP2 and exert similar antifungal activity in vitro.
The cDNA encoding Rs-AFP1 encodes a preprotein with a signal peptide followed by the mature protein. The cDNA sequence is shown in
FIG. 2.
Saccharomyces cerevisiae
can be used as a vector for the production and secretion of Rs-AFP2 (Vilas Alves et al, FEBS Lett, 1994, 348:228-232). Plant-derivable “wild-type” Rs-AFP2 can be correctly processed and secreted by yeast when expressed as a N-terminal fusion to the yeast mating factor &agr;1 (MF&agr;1) preprosequence. The Rs-AFP2 protein does not have adverse effects on yeast even at concentrations as high as 500 &mgr;g/ml.
We now provide new potent antifungal peptides based on the structure of the Rs-AFPs and related plant defensins.
According to the first aspect of the present invention there is provided an antifungal peptide which comprises at least six amino acid residues identical to a run of amino acid residues found between position 21 and position 51 of the Rs-AFP2 sequence shown in
FIG. 1
or of substantially homologous protein sequences.
Proteins which are substantially homologous to the Rs-AFP2 protein include the proteins Rs-AFP1, Rs-AFP3, Rs-AFP4, Br-AFP1, Br-AFP2, Bn-AFP1, Bn-AFP2, Sa-AFP1, Sa-AFP2 and At-AFP1 shown in FIG.
1
and Hs-AFP2, Ah-AMP1 and Dm-AMP1 shown in FIG.
9
. Proteins which are substantially homologous have an amino acid sequence with at least 40% sequence identity to any of the sequences shown in
FIGS. 1 and 9
, and preferably at least 60% identity; and most preferably at least 80% identity.
Antifungal peptides according to the invention include especially peptides derived from the beta-2 strand/turn/beta-3 strand region of Rs-AFP2 and substantially homologous antifungal protein sequences. Preferred antifungal peptides according to the invention include the 6-mer, 9-mer and 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer and 20-mer, and most especially the 18-mer, 19-mer and 20-mer peptides described in the accompanying examples, figures and tables especially Example 11 and
FIGS. 10
to
13
.
Antifungal peptides according to the invention include the following peptides:
a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 21 and position 35 of the Rs-AFP2 sequence shown in FIG.
1
and having the sequence: CKNQCIRLEKARHGS;
a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 25 and position 39 of the Rs-AFP2 sequence shown in FIG.
1
and having the sequence: CIRLEKARHGSCNYV;
a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 29 and position 43 of the Rs-AFP2 sequence shown in FIG.
1
and having the sequence: EKARHGSCNYVFPAH;
a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 33 and position 47 of the Rs-AFP2 sequence shown in FIG.
1
and having the sequence: HGSCNYVFPAHKCIC;
a peptide comprising ten amino acid residues identical to a run of ten amino acid residues found between position 36 and position 45 of the Rs-AFP2 sequence shown in FIG.
1
and having the sequence: CNYVFPAHKC;
a peptide comprising six amino acid residues identical to a run of six
Borremans Frans Alois
Broekaert Willem Frans
De Samblanx Genoveva Wivina
Fant Franky
Meloen Robbert Hans
Hale & Dorr
Low Christopher S. F.
Robinson Hope A.
Syngenta Limited
Syngenta Limited
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