Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Reexamination Certificate
1999-05-06
2001-06-05
Bui, Phuong T. (Department: 1638)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
C435S069100, C435S070100, C435S071100, C435S071200, C435S410000, C435S418000, C435S419000, C530S300000, C530S324000, C530S379000, C530S370000, C536S023600, C800S279000, C424S195110
Reexamination Certificate
active
06242574
ABSTRACT:
The present invention relates to anti-microbial proteins isolatable from sugar beet.
According to the present invention there is provided anti-microbial protein comprising a peptide having the sequence -Gln/Cys-AA
2
-Pro/Ile-Asn/Thr/Leu-AA
5
-AA-Cys-Cys-Ala/Asn-Gly/Lys-AA
11
-AA
12
-AA
13
-AA
14
-AA
15
-(SEQ ID NO: 35), with the proviso that AA
2
and AA
14
are not cysteine, and that AA
4
is Leu when AA
1
is Cys. The skilled man will recognize that the abbreviation AA
x
means one of the 20 commonly occurring amino acids.
An anti-microbial protein includes a protein (alone or in combination with another material) which is toxic or growth inhibitory under any circumstances to any micro-organism, including bacteria, (most particularly Gram positive bacteria), viruses and particularly fungi. Such anti-microbial proteins include those that exhibit anti-microbial activity upon contact with a micro-organism and those that are anti-microbial as a consequence of assimilation or respiration thereof.
According to the present invention there is provided an anti-microbial protein having the sequence depicted in any one of SEQ ID Nos. 3, 5 or 6.
Also included is an isoform of an anti-microbial protein having SEQ ID No. 3 wherein Val in pos. 80 is Ala.
The invention still further includes pure protein which is substantially similar to any one of the above mentioned proteins.
By “substantially similar” is meant pure proteins comprising an amino acid sequence which ist at least 95% similar to the peptide sequence given above (also as defined in claim
1
) and/or pure proteins having an amino acid sequence which is at least 65% similar, preferably 75% similar, more preferred 85% similar, particularly preferred 95% similar to the sequence of the proteins depicted in SEQ ID Nos 3, 5 or 6 below. In the context of the present invention, two amino acid sequences with a given percentage similarity to each other have at least that percentage of identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 2 gaps with the proviso that in respect of each gap a total not more than 2 amino acid residues is affected.
For the purpose of the present invention conservative replacements may be made between amino acids within the following groups:
(i) Serine and Threonine;
(ii) Glutamic acid and Aspartic acid;
(iii) Arginine and Lysine;
(iv) Asparagine and Glutamine;
(v) Isoleucine, Leucine, Valine and Methionine;
(vi) Phenylalanine, Tyrosine and Tryptophan
(vii) Alanine and Glycine
The invention still further includes pure proteins which are at least 90% identical to the anti-microbial proteins according to the invention, as well as pure proteins which have at least 90% of the specific activity thereof. For the purposes of the present application, specific activity is a measurement of the amount of growth or replication inhibition produced by a specified quantity of the protein on a specified quantity of a specified micro-organism.
The invention still further includes said pure proteins in combination with at least one protein selected from the group consisting of those depicted in SEQ ID Nos. 7 to 12 and 14. Such combined proteins may be further combined with one or more of the known “pathogenesis-related proteins”. Infection of plants with fungal or viral pathogens may induce a systemic synthesis of about 10 families of homologous pathogenesis-related proteins (PR proteins) in vegetative tissues. Such PR-proteins have been classified into 5 groups. The PR-2, PR-3 and PR-5 proteins are beta-1,3-glucanase, chitinases and thaumatin-like proteins respectively. Specific functions have not been assigned to the PR-1 and PR-4 groups of proteins. The PR4 proteins are similar to C-terminal domains of prohevein and the putative wound-induced WIN proteins of potato, thus lacking the N-terminal hevein domain. It is particularly preferred that the proteins according to the invention are combined with one or more proteins which are the basic counter parts of the P-R 4 group of proteins, meaning the basic counter part of proteins similar to the C-terminal domains of prohevein and the putative wound-induced WIN proteins of potato. It is particularly preferred that the basic counter-part of the said pathogenesis-related proteins is a chitin-binding WIN protein, in particular that produced by barley grain or stressed barley leaves.
The invention still further includes recombinant DNA comprising a sequence encoding a protein having the amino acid sequence of the above disclosed anti-microbial proteins. In particular the DNA may encode at least one of the proteins the sequences of which are depicted in SEQ ID Nos. 3, 5 and 6, optionally in addition to at least one of the proteins the sequences of which are depicted in SEQ ID Nos. 7 to 12 and 14. Preferably the sequence encoding a protein having the amino acid sequence depicted in SEQ ID No. 3, 5 or 6 has the cDNA sequence depicted in SEQ ID No. 1 (for the protein with SEQ ID No. 6, corresponding to IWF5), in SEQ ID No. 2 (for the protein with SEQ ID No. 3, corresponding to IWF6) or in SEQ ID No. 4 (for the protein with SEQ ID No. 5, corresponding to IWF7). The recombinant DNA may further encode a protein having herbicide resistance, plant growth-promoting, anti-fungal, anti bacterial, anti-viral and/or anti-nematode properties. In the case that the DNA is to be introduced into a heterologous organism it may be modified to remove known mRNA instability motifs (such as AT-rich regions) and polyadenylation signals (if any are present). and/or codons which are preferred by the organism into which the recombinant DNA is to be inserted may be used so that expression of the thus modified DNA in the said organism yields substantially similar protein to that obtained by expression of the unmodified recombinant DNA in the organism in which the anti-microbial protein according to the invention is endogenous.
The invention still further includes recombinant DNA which is “similar” to that mentioned above. By “similar DNA” is meant a sequence which is complementary to a test sequence which is capable of hybridizing to the inventive recombinant sequence. When the test and inventive sequences are double stranded the nucleic acid constituting the test sequence preferably has a TM within 20° C. of that of the inventive sequence. In the case that the test and inventive sequences are mixed together and denatured simultaneously, the TM values of the sequences are preferably within 10° C. of each other. More preferably the hybridization is performed under stringent conditions, with either the test or inventive DNA preferably being supported. Thus either a denatured test or inventive sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 50 and 70° C. in double strength citrate buffered saline (SSC) containing 0.1% SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, at a particular temperature,—such as 60° C., for example—such reduced concentration buffers are typically single strength SSC containing 0.1% SDS, half strength SSC containing 0.1% SDS and one tenth strength SSC containing 0.1% SDS. Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the test and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing 0.1% SDS.
The invention still further includes a DNA sequence which is complementary to one which hybridizes under stringent conditions with the recombinant DNA according to the invention.
Also included in the present invention is: a vector which contains the above disclosed DNA which is expressible in plants and linked to a plant operable pr
Brunstedt Janne
Kroll Kristensen Anne
Nielsen Klaus Kristian
Bui Phuong T.
Meigs J. Timothy
Syngenta Participations (AG)
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