Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-03-11
2002-04-16
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S300000, C530S324000, C800S301000, C800S302000, C800S281000, C435S007200, C435S007310, C435S320100, C435S173300, C435S419000, C514S012200, C536S023600
Reexamination Certificate
active
06372888
ABSTRACT:
FIELD OF INVENTION
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.
BACKGROUND
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 WO93/05153 published Mar. 18, 1993; Terras FRG et al, 1992, J Biol Chem, 267:15301-15309; Terras et al, FEBS Lett, 1993, 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 as plant defensins (Terras F. R. G. et al 1995, Plant Cell 7 573-588) and these proteins share a similar motif of conserved cysteines and glycines (Broekaert et al 1995 Plant Physiol 108 1353-1358).
FIG. 1
shows the amino acid sequences of the protein Rs-AFP2 (SEQ ID NO: 9) and the substantially homologous proteins Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10), Rs-AFP4 (SEQ ID NO: 11), Br-AFP1 (SEQ ID NO: 12), Br-AFP2 (SEQ ID NO: 13), Bn-AFP1 (SEQ ID NO: 14), Bn-AFP2 (SEQ ID NO: 15), Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2 (SEQ ID NO: 17) and At-AFP1 (SEQ ID NO: 18) 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 sequence indicates a gap introduced for maximum alignment. The sequences shown for Br-AFP1, Br-AFP2, Bn-AFP1, Bn-AFP2, Sa-AFP1, Sa-AFP2 and At-AFP1 are not complete: only the N-terminal sequences are shown. The question mark (?) in the Br-AFP2 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.
The primary structures of the two antifungal protein isoforms capable of isolation from radish seeds, Rs-AFP1 (SEQ ID NO: 8) and Rs-AFP2 (SEQ ID NO: 9), only differ at two positions: the glutamic acid residue (E) at position 5 in Rs-AFP1 (SEQ ID NO: 8) is a glutamine residue (Q) in Rs-AFP2 (SEQ ID NO: 9), and the asparagine residue (N) at position 27 in Rs-AFP1 (SEQ ID NO: 8) is substituted by an arginine residue (R) in Rs-AFP2 (SEQ ID NO: 9). As a result, Rs-AFP2 (SEQ ID NO: 9) 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 (SEQ ID NO: 9) is two- to thirty-fold more active than Rs-AFP1 (SEQ ID NO: 8) on various fungi and shows an increased salt-tolerence. The proteins Rs-AFP3 (SEQ ID NO: 10) and Rs-AFP4 (SEQ ID NO: 11) are found in radish leaves following localized fungal infection. The induced leaf proteins are homologous to Rs-AFP1 (SEQ ID NO: 8) and Rs-AFP2 (SEQ ID NO: 9) and exert similar antifungal activity in vitro.
The cDNA encoding Rs-AFP1 (SEQ ID NO: 19) encodes a preprotein with a signal peptide followed by the mature protein. The cDNA sequence is shown n
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 proteins based on the structure of the Rs-AFPs and related proteins.
SUMMARY OF THE INVENTION
According to a first aspect the invention provides an antifungal protein having an amino acid sequence which is substantially homologous to the Rs-AFP2 sequence (SEQ ID NO: 9) shown in FIG.
1
and containing at least one mutation selected from the group consisting of a basic residue at position 9, a basic residue at position 39, a hydrophobic residue at position 5 and a hydrophobic residue at position 16.
According to a preferred embodiment of the first aspect of the present invention there is provided an antifungal protein having an amino acid sequence which is substantially homologous to the Rs-AFP2 sequence (SEQ ID NO: 9) shown in FIG.
1
and containing at least one mutation selected from the group consisting of an arginine residue at position 9, an arginine residue at position 39, a methionine residue at position 5 and a methionine residue at position 16. An antifungal protein having both a mutation to arginine at position 9 and a mutation to arginine at position 39 may be particularly active.
Proteins which are substantially homologous to the Rs-AFP2 protein include the proteins Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10), Rs-AFP4 (SEQ ID NO: 11), Br-AFP1 (SEQ ID NO: 12), Br-AFP2 (SEQ ID NO: 13), Bn-AFP1 (SEQ ID NO: 14), Bn-AFP2 (SEQ ID NO: 15), Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2 (SEQ ID NO: 17) and At-AFP1 (SEQ ID NO: 18) shown in FIG.
1
.
As used herein the term substantially homologous denotes those proteins which have an amino acid sequence with at least 40% identity, preferably at least 60% identity and most preferably at least 80% identity to the Rs-AFP2 sequence (SEQ ID NO 9).
The invention further provides an antifungal peptide which comprises at least six amino acid residues identical to a run of amino acid residues in an antifungal protein according to the invention, said run of residues including at least one of the mutated residues.
In particular, there are provided the following antifungal proteins and antifungal peptides derived therefrom:
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), Rs-AFP2 (SEQ ID NO: 9), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which the glycine residue at position 9 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), Rs-AFP2 (SEQ ID NO: 9) or Rs-AFP3 (SEQ ID NO: 10) in which the valine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP4 (SEQ ID NO: 11) in which the isoleucine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-APF1 (SEQ ID NO: 8), Rs-AFP2 (SEQ ID NO: 9) or Rs-AFP3 (SEQ ID NO: 10) in which the glycine residue at position 9 is replaced by an arginine residue and the valine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP4 (SEQ ID NO: 11) in which the glycine residue at position 9 is replaced by an arginine residue and the isoleucine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which the glutamic acid residue at position 5 is replaced by a methionine residue;
a protein having the amino acid sequence of Rs-AFP2 (SEQ ID NO: 9)in which the glutamine residue at position 5 is replaced by a methionine residue;
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), RS-AFP2 (SEQ ID NO: 9), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which
Broekaert Willem Frans
De Samblanx Genoveva Wivina
Rees Sarah Bronwen
Carlson Karen Cochrane
Hale and Dorr LLP
Robinson Hope A.
Zeneca Limited
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