Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1997-11-17
2001-02-13
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S326000, C530S350000, C514S012200, C514S013800
Reexamination Certificate
active
06187904
ABSTRACT:
This invention relates to biocidal proteins, processes for their manufacture and use, and DNA sequences coding for them. In particular, it relates to antimicrobial proteins isolated from seeds such as those of members of the Brassicaceae, Compositae or Leguminosae families.
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); antibacterial activity. Activity includes a range of antagonistic effects such as partial inhibition or death.
The Brassicaceae is a large family of herbs and shrubs which grow widely in tropical, sub-tropical and temperate regions. The Family Brassicaceae is also known as the “Cruciferae”.
Raphanus sativus
(radish) belongs to this family and is cultivated widely as a vegetable.
Dahlia belongs to the Compositae and has been extensively cultivated as an ornamental garden plant. A number of hybrids are commercially available, belonging to the
Dahlia merckii
or
Dahlia variablis
species.
Cnicus benedictus
, another Compositae, is a native plant of the Mediterranean regions and was once used as a tonic and a cure for gout.
Lathyrus and Clitoria belong to the Leguminosae family. Lathyrus has been extensively cultivated as an ornamental garden plant, the most widely known being the sweet pea plant, Lathyrus odoratus. The genus Clitoria is less well known to European gardeners;
Clitoria ternatea
was originally introduced from the East Indies in the 1800s.
Although plants normally grow on substrates that are extremely rich in fungal organisms, infection remains a rare event. To keep out potential invaders, plants produce a wide array of antifungal compounds, either in a constitutive or an inducible manner. The best studied of these are phytoalexins which are secondary metabolites with a broad antimicrobial activity spectrum that are specifically synthesised upon perception of appropriate defence-related signal molecules. The production of phytoalexins depends on the transcriptional activation of a series of genes encoding enzymes of the phytoalexin biosynthetic pathway. During the last decade, however, it has become increasingly clear that some plant proteins can play a more direct role in the control of phytopathogenic fungi. Several classes of proteins with antifungal properties have now been identified, including chitinases, beta-1,3-glucanases, chitin-binding lectins, zeamatins, thionins and ribosome-inactivating proteins.
These proteins have gained considerable attention as they could potentially be used as biocontrol agents. The chitinases and beta-1,3-glucanases have weak activities by themselves, and are only inhibitory to plant pathogens when applied in combination (Mauch et al, 1988, Plant Physiol, 88, 936-942). The chitin-binding lectins can also be classified as rather weak antifungal factors (Broekaert et al, 1989, Science, 245, 1100-1102; Van Parijs et al, 1991, Planta, 183, 258-264). Zeamatin is a more potent antifungal protein but its activity is strongly reduced by the presence of ions at physiological concentrations (Roberts and Selitnermikoff, 1990, G Gen Microbiol, 136, 2150-2155). Finally, thionins and ribosome-inactivating proteins are potentially hazardous since they are known to be toxic for human cells (Carrasco et al, 1981, Eur J Biochem, 116, 185-189; Vernon et al, 1985, Arch Biochem Biophys, 238, 18-29; Stirpe and Barbieri, 1986, FEBS Lett, 195, 1-8).
We have now purified a new class of potent antimicrobial proteins with broad spectrum activity against plant pathogenic fungi and with some antibacterial activity, moderate sensitivity to ions and apparent low toxicity for cultured human cells.
According to the present invention, we provide antimicrobial proteins capable of being isolated from seeds and in particular from members of the Brassicaceae, the Compositae or the Leguminosae families including Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus or Clitoria.
In further aspects, this invention comprises a vector containing a DNA sequence coding for a protein according to the invention. The DNA may be cloned or transformed into a biological system allowing expression of the encoded protein.
The invention also comprises plants transformed with recombinant DNA encoding an antimicrobial protein according to the invention.
The invention also comprises a process of combating fungi or bacteria whereby they are exposed to the proteins according to the invention.
A new class of potent antimicrobial proteins has been isolated from seeds of the Brassicaceae, the Compositae, and the Leguminosae. Similar proteins may be found in other plant families, genera and species. The class includes proteins which share a common amino acid sequence and which show activity against a range of plant pathogenic fungi.
The antimicrobial proteins isolated from seeds of
Raphanus sativus
(radish) include two protein factors, hereafter called Rs-AFP1 (
Raphanus sativus
—Antifungal Protein 1) and Rs-AFP2 (
Raphanus sativus
—Antifungal Protein 2) respectively. Both are oligomeric proteins, composed of identical 5 kDa subunits. Both proteins are highly basic and have pI values above 10. Similar antifungal proteins have been isolated from other Brassicaceae, including
Brassica napus
(Bn-AFPs),
Brassica rapa
(Br-AFPs),
Sinapis alba
(Sa-AFPs) and
Arabidopsis thaliana
(At-AFP1).
The antimicrobial proteins isolated from seeds of Dahlia and Cnicus include four protein factors, hereafter called Dm-AMP1 (
Dahlia merckii
—Antimicrobial Protein 1), Dm-AMP2 (
Dahlia merckii
—Antimicrobial Protein 2), Cb-AMP1 (
Cnicus benedictus
—Antimicrobial Protein 1) and Cb-AMP2 (
Cnicus benedictus
—Antimicrobial Protein 2) respectively. The Dm-AMP proteins may be isolated from seed of the Dahlia genus. The Cb-AMP proteins may be isolated from seed of the Cnicus genus. All four proteins are closely related and are composed of 5 kDa subunits arranged as oligomeric structures. All four proteins are highly basic.
The antimicrobial proteins isolated from seeds of Lathyrus and Clitoria include three protein factors, hereafter called Lc-AFP (
Lathyrus cicera
—Antifungal Protein), Ct-AMP1 (
Clitoria ternatea
—Antimicrobial Protein 1) and Ct-AMP2 (
Clitoria ternatea
—Antimicrobial Protein 2) respectively. Lc-AFP may be isolated from seed of the Lathyrus genus. The Ct-AMP proteins may be isolated from seed of the Clitoria genus. All three proteins are composed of 5 kDa subunits arranged as oligomeric structures and are highly basic.
N-terminal amino acid sequence determination has shown that the above proteins isolated from the Brassicaceae, Compositae and Leguminosae are closely related and can be classified as a single protein family. Between the different plant families, the protein sequences are approximately 50% identical. These sequences enable manufacture of the proteins by chemical synthesis using a standard peptide synthesiser.
The antimicrobial proteins are partially homologous to the predicted protein products of the Fusarium-induced genes pI39 and pI230 in pea (
Pisum sativum
—a member of the Leguminosae family) as described by Chiang and Hadwiger, 1991 (Mol Plant Microbe Interact, 4, 324-331). This homology is shared with the predicted protein product of the pSAS10 gene from cowpea (
Vigna unguiculata
—another legume) as described by Ishibashi et al (Plant Mol Biol, 1990, 15, 59-64). The antimicrobial proteins are also partially homologous with the predicted protein product of gene pI322 in potato (
Solanum tuberosum
—a member of the Solanaceae family) as described by Stiekema et al, 1988 (Plant Mol Biol, 11, 255-269). Nothing is known about the biological properties of the proteins encoded by genes pI39, pI230, pSAS10 or pI322 as only the CDNA has been studied. However, the pI39, pI230 and pI322 genes are switched on after challenge to the plant by a disease or other stress. It has been proposed that the pSAS10 gene encodes a protein involved in germination. Due to their sequence similarity with the antimicrobial proteins of the i
Broekaert Willem F.
Cammue Bruno P. A.
Osborn Rupert W.
Rees Sarah B.
Terras Franky R. G.
Gupta Anish
Hohenschutz Liza D.
Low Christopher S. F.
Zeneca Limited
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