Antimicrobial proteins

 

(57) Abstract:

The invention relates to the production of antimicrobial proteins that are used to control fungi. Proteins isolated from sugar beet and have the amino acid sequence represented in any of the sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. The obtained recombinant DNA encoding the above proteins and having the sequence represented in SEQ ID NO 7. The above proteins have antifungal activity against fungi species C. beticola. 3 S. and 1 C.p. f-crystals, 9 Il.

The object of the present invention are antimicrobial proteins secreted from sugar beets.

According to the present invention offer antimicrobial protein containing a peptide having the following amino acid sequence: AA1-AA2-AA3-Cys-AA5-AA6-AA7-AA8-AA9-Cys-AA11- AA12-AA13-AA14-Cys-Cys-AA17-AA18- AA19-AA20-AA21-Cys-AA23-AA24-AA25-AA26-AA27-AA28-Cys-AA30where "AA" means any of the known 20 amino acids. Preferably, AA7was tyrosine; AA14was tyrosine; AA18was lysine. Chrome is vslas by alanine.

Under antimicrobial protein mean protein (alone or in combination with other material) that is toxic or inhibiting the growth in any circumstances of any microorganism, including bacteria (most often gram-positive bacteria), viruses, and especially mushrooms. Among these antimicrobial proteins are proteins that exhibit antimicrobial activity upon contact with the microorganism, as well as proteins, antimicrobial properties which are the result of their accumulation or inhalation.

The invention also includes an antimicrobial protein having the sequence described in any of the PEFC. N 1-3.

Further, the invention includes pure protein which is substantially similar to any of the above proteins.

The term "substantially similar" understand pure protein having the amino acid sequence that is at least 85% similar to the sequence of the protein according to the invention. Preferably, the degree of similarity was at least 90%, even more preferably, the degree of similarity was at least 95%.

In the context of the present invention, two amino acid sequences with at least 85%, 90% minocycline residues in the same position at the optimum location, allow up to 2 breaks provided that in respect of each gap involved in General not more than 3 amino acid residue. In the case of proteins, specifically described in Th. N is 1 or 2, the number of breaks can be increased up to 4, provided that in respect of each gap involved in General not more than 5 amino acid residues.

For the purposes of the present invention can be produced conservative substitutions between amino acids within the following groups:

(I) serine and Threonine;

(II) Glutamic acid and Aspartic acid;

(III) Arginine and Lysine;

(IV) the Asparagine and Glutamine;

(V) Isoleucine, Leucine, Valine and Methionine;

(VI) Phenylalanine, Tyrosine and Tryptophan;

(VII) Alanine and Glycine.

Further, the invention includes pure proteins that are at least 90% identical antimicrobial protein according to the invention, and clean proteins that have at least 90% of their specific activity. From the point of view of the purposes of the present application under specific activity understand the quantitative measurement of inhibiting the growth or replication caused a certain amount of protein in relation to a specific number is definitely the necks least one protein, selected from the group consisting of proteins, are described in Th. N 4-6. Such combined proteins can be further combined with one or more of known progressivism proteins". Infection of plants, fungal, or viral pathogens can be induced in plant tissues systemic synthesis of more than 10 families of homologous progressivism proteins (PZ-proteins). These PZ-proteins were classified into 5 groups. PZ-2, PZ-3 and PZ-5 proteins are beta-1,3-glucanases, chitinases and DomainPeople proteins, respectively. PZ-1 and PZ-4-groups of proteins have not been assigned specific functions. PZ-4-similar proteins with C-terminal domains prohevein and presumably induced by wounding a game of PIN proteins potatoes, not having, therefore, N-terminal domain Geneina. Most preferably, the proteins according to the invention were combined with one or more proteins, which are the main opposing parts of the PZ-4-group proteins, implying opposite the main part of the protein, similar to C-terminal domains prohevein and presumably induced by wounding a game of PIN proteins potatoes. Most preferably, opposite the main part, these progressivism proteins allergologia stressful effects.

Further, the invention includes a recombinant DNA containing a sequence encoding a protein having the amino acid sequence of the above-described antimicrobial proteins. In particular, the DNA may encode at least one protein, the sequence of which is described in Th. N 1-3, perhaps in addition to at least one of the proteins, the sequence of which is described in Th. N 4-6. In addition, the recombinant DNA can encode a protein having the properties of herbicidetolerant, stimulation of plant growth, antigribnaya, antibacterial, antiviral and/or antisemitisme properties. In the case when DNA must be introduced into a heterologous organism, it can be modified by removing the known motifs mRNA instability (such as at-rich region) and polyadenylation signals (if present), in addition, the codons that are preferred by the body, which must be administered recombinant DNA, can be used so that expression of such a modified DNA in the specified organism led to the formation of a protein substantially similar to the protein, which is obtained by expression of the unmodified Ryoko is

In addition, the invention includes a recombinant DNA which is "similar" DNA mentioned above. Under "similar DNA" means a sequence that is complementary to the test sequence, which is capable of hybridization with the proposed recombinant sequence. In the case when the test sequence and the proposed sequence are Dantewada, a nucleic acid that consists of a test sequence, preferably has a TPLdiffer no more than 20oC from the values of TPLthe proposed sequence. In the case when the test sequence and the proposed sequence are mixed together and are denatured simultaneously, the values of TPLsequences preferably differ from each other not more than 10oC. More preferably to hybridization under strict conditions, when the test DNA, or offer DNA preferably located on the substrate. Then, either a denatured test sequence or the proposed sequence is preferably first bound to the substrate, and the hybridization is carried out in a certain period of time at a temperature between 50 and of the substrate at the same temperature, but a buffer having a reduced concentration DSC. Depending on the required degree of accuracy, and hence the degree of similarity between the sequences of these buffers with a low concentration are usually single DSC containing 0.1% DDS, DSC with half concentration, containing 0.1% VAT, and DSC concentration equal to one tenth of the original, containing 0.1% VAT. The sequences with the greatest degree of similarity, are sequences, hybridization which are least affected by washing in buffers of low concentration. Most preferably, the test sequence and the proposed sequence was so similar that the hybridization between them was not materially affected by washing or incubation in sodium citrate buffer concentration equal to one tenth of the original, containing 0.1% VAT.

Further, the invention includes a DNA sequence that is complementary to the sequence, which hybridizes in stringent conditions with the recombinant DNA according to the invention.

In addition, the present invention includes: a vector that contains the above-described DNA, which can be expressed in plants and is associated with destiny, which contains DNA stably integrated and inherited according to the law of Mendel, and/or seeds of such plants and the offspring. Transgenic plants obtained by known methods, which include the regeneration of plant cells or protoplasts transformed with DNA according to the invention according to various known methods (Ti and Ri plasmids of Agrobacterium, electroporation, microinjection, mikrotalasna gun and so on). Transformed cell in the respective cases can be regenerated into whole plants, in which nuclear material is stably incorporated into the genome. Thus can be obtained as monocotyledonous and dicotyledonous plants. Examples of the transformed plants according to the present invention include: fruits, including tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons; field crops such as canola, sunflower, tobacco, sugar beets, cereals such as wheat, barley and rice, corn and cotton, and vegetables, such as potatoes, carrots, lettuce, cabbage and onion. Preferred plants are sugar beets, and corn.

Further, the invention includes a protein produced by the expression of the indicated DNA, and anti-microbial white>Further, the invention includes an antimicrobial composition containing one or more proteins according to the invention; a method of combating fungi, which includes the processing of such proteins, and methods of extraction to produce antimicrobial proteins from organic material containing it, including the handling of the material is preferably in the form of the organism through maceration and solvent extraction. It should be noted that the antimicrobial protein has a small, if at all, an antimicrobial effect on the organism that is the source of organic material, which was mentioned in the previous paragraph.

Hereinafter the invention will be detailed based on the following description and accompanied drawings and sequence listings.

In Fig. 1 shows a typical elution profile of intracellular washing liquid from the column with KM-Separate;

in Fig. 2 shows the characteristic profile of elution from the column Mono S JAHB 0.3 M NaCl fraction, shown in Fig. 1;

in Fig. 3 shows the characteristic profile of elution from the column OF GHUR proteins, detected in the form of peak 3 in Fig. 2;

in Fig. 4 shows antigena activity 10 ug of protein, detectional on Sephadex G-75 0.1 M fraction, presented on Fig. 1;

in Fig. 6 shows the characteristic profile of elution from the column Mono S JAHB peak (pool) V, shown in Fig. 5;

in Fig. 7 shows antigena activity 10 ug of protein, detected as peaks 1 and 2 (Th. N 1) in Fig. 6;

in Fig. 8A shows the combined antigena activity 2 ug of each protein is presented Last. N 2 and 3;

in Fig. 8B shows the combined antimicrobial activity of 2 ug of each protein is presented Last. N 2 and 5;

in Fig. 9A and 9B shows the morphology of the hyphae of C. beticola, grown in the absence of antimicrobial protein according to the invention;

in Fig. 9C and 9D shows hyphae in the case when the fungus grows within 48 hours in the presence of 2 ug of protein, having the sequence described in Th. N 2, and 3, respectively.

The experience carried out in microtiter tablets, as described below; the increase in Fig. 9A - 76X; Fig. 9B-9D - 294X.

PEFC. N 1 represents the amino acid sequence of the protein, detected as a peak of 3.1 in Fig. 3; Th.N 2 is the amino acid sequence of the protein, detected as a peak of 3.2 in Fig. 3; and PEFC. No. 3 shows the amino acid sequence of the protein, detected as peaks 1 and 2 in Fig. 6; Th. N 4-6 positivedefinite cDNA, encodes a protein described in Th. N 3; and PEFC. N 8 shows the product of the translation of the transcript encoded by the cDNA sequence described in Th. N 7; this product contains N - and C-terminal extension segments of the protein described in Th. N 3.

The allocation of intercellular washing fluid

VPI separated from 500-700 grams of sugar beet leaves by immersion in 20 mm NAC (pH 4.5). Shipped so the leaves were then placed in a desiccator and subjected to vacuum filtration for 5 minutes at 4 Torr (maximum). After air drying the surface of the leaves VPI collected by centrifugation at 500 g for 15 minutes in the centrifuge tubes with a capacity of 500 ml.

Cation-exchange chromatography

Thus obtained VPI fractionary by cation exchange chromatography on a column with KM-Separate capacity of 10 ml (Pharmacia LKB) that was previously equilibrated with the starting buffer (20 mm NAC (pH 4.5)). The fractionation is carried out at 4oC when the velocity of the flow 25 ml/year Collect fractions of 3 ml of Proteins, not contacting column, is removed by thorough washing of the column with the starting buffer. Associated proteins elute by drawing on additional column starting benie of the eluate at 280 nm, and fractions, presumably containing the protein are tested for their anthropou activity against C. beticola way to vioience in microtiter tablets, as described previously [1].

A typical elution profile is shown in Fig. 1. Eluate, resulting after application to the column of the starting buffer containing 0.3 M NaCl and 0.1 M NaCl, purified further as described below.

Cleaning antigenic proteins in 0.3 M NaCl the eluate obtained after JAHB chromatography on KM-Sepharose

0.3 M NaCl protein fraction absoluut by dialysis during the night (MV: 3 KD) against 20 mm NAC (pH 4.5) at 4oC. To detalizirovannoi thus protein fraction add betaine at a concentration of 5%. Four ml of the resulting solution fractionary then by cation exchange liquid Express chromatography of proteins (IAHB) using a Mono S HR 5/5 column (Pharmacia LKB), equilibrated in 20 mm NAC (pH 4.5) containing 5% (V/V) betaine (A-buffer). Associated proteins elute with a linear salt gradient from 0 to 0.3 M NaCl in 30 ml of A buffer with the subsequent stage by elution with 1.0 M NaCl in the same buffer. The flow rate is 1 ml/min.

Fig. 2 shows that the 0.3 M NaCl fraction contains a number of individual proteins, quantitatively the most significant of which I have (Pharmacia LKB) painted silver 10-15% gradient Phast gels or gels with high density (Pharmacia LKB) installed, that the peaks 1-5 contain 2-5 protein bands.

GHUR with reversed phase

The protein peak 3 (indicated in Fig. 2) from the Mono S column purified further using GHUR with reversed phase (RP-) on a Vydac C4the column of silicon (The Separations Group, CA, USA). The solvent system is system a: 0.1% TN in water and B: 0.1% TN in acetonitrile. Proteins elute with a linear gradient from 5 to 45% B-buffer, applied after 18 minutes after application of the sample, followed by the application 60% B-buffer after 2 minutes. The flow rate 0.7 ml/min Protein identified by registration of the eluate absorbance at 214 and 280 nm. Individual protein peaks collected and lyophilizers. Lyophilized thus proteins are then washed twice with water, re-lyophilizer and then dissolved in 10 mm Tris-HCl (pH 8.0) prior to analysis for purity and anthropou activity.

Fig. 3 shows that the peak 3 of Fig. 2 is divided into four peak (labeled 3.1-3.4 in Fig. 3) on the PF column. DDS-PAG peaks 3.1-3.4 shows that each of them consists of four proteins having a molecular weight of about 7 KD (peaks 3.1, 3.2 and 3.3) and 2.5-3 CD (3.4).

Cleaning antigenic proteins of 0.1 M of NaCl eluate from KM-Sepharose

Five ml of 0.1 M of NaCl eluate from Fig. 1 fractionary Geel the current 20 ml/H. Collect fractions with a volume of 10 ml. These fractions are sequentially divided into six large fractions I-VI, containing approximately 50 ml each (Fig. 5).

Cation exchange (Mono S)

Pool V (shown in Fig. 5) from the column with Sephadex G-75 is applied to a Mono S JAHB column, equilibrated with buffer A: 50 mm MES (pH of 6.0) containing 5% betaine (/about). After washing And buffer bound peroxidase proteins elute at a speed of flow of 1 ml/min linear gradient from 0 to 0.5 M NaCl in 30 ml of a buffer (Fig. 6). When DDS-gel electrophoresis in the presence of DTT protein, detected as peaks 1 and 2, see two separate lanes. The first band has a molecular weight of between 2.5 and 3 KD, and the second band, which is deregulirovanye dimer protein first strip has a molecular weight of about 5 KD.

IDENTIFICATION ANTIGENIC PROTEIN

Antigena activity

Fig. 4 and 7-9 show that proteins, detected as peaks 3.1 and 3.2 (PEFC. N 1 and 2, respectively) in Fig. 3, and peaks 1 and 2 (Th. N 3) in Fig. 6 have significant anthropou activity, among other things, against C. beticola. Inhibition of fungal growth was measured in 96-well microtiter-plate at 620 nm as described [1]. Fig. 9 shows that C. beticola treated t is to fungus, growing in the absence of antimicrobial proteins (Fig. 9A, 9B).

Proteins, alone or in combination with PIN N (which is separated from the grains of barley or barley leaves subjected to stressful effects as described [2]), and/or protein having a sequence corresponding to at least one of the PEFC. N 4-6, incubated with spores of C. beticola. Experimental mixture (240 ul) contains 100 str potato agar with glucose (Difco), 40 ul of protein sample (or buffer control) in 100 mm Tris and 20 mm NaCl (pH 8.0), and approximately 400 spores in 100 ul of water. Microtiter tablets stick tape to avoid evaporation and contamination, and then incubated at room temperature on a shaker operating at 200 rpm Daily for 8 days measure the absorption at 620 nm and build the graphical dependence of the concentration of protein from time to time.

Amino acid sequencing

Peeled antigene proteins corresponding to the peaks 3.1 and 3.2 in Fig. 3 (Th. N 1 and 2, respectively), the source of which is 0.3 M NaCl eluate from the column with KM-Separate (Fig. 1); and protein, corresponding to peaks 1 and 2 in Fig. 6 (Th. N 3), the source of which is 0.1 M NaCl eluate from this column with KM-Separate, carboxylic and under the B: 0.1% TN in acetonitrile. Proteins suiryudan as separate peaks with maroulakis values of retention time. C-terminal sequences of the proteins get by splitting their endo-R-proteinase and subsequent purification by means OF GHUR on a Vydac C18column.

Obtaining transformed plants

The genes encoding the proteins according to the invention, is introduced into plants. Based genespecific primers from the corresponding mRNA by PCR synthesize coding region of the genes encoding these proteins. After the introduction of the promoter and termination sequences of the genes encoding these proteins are injected into a vector for transformation of plants. The vector can include the gene encoding the PIN protein, and protein derived from the leaf of barley subjected to stressful effects, or a grain of barley, and/or the gene encoding the protein described in Th. N 4, Th. N 5 and/or PEFC. N 6, and/or the gene encoding the chitinase and/or glucanase. The preferred chitinase chitinase is 4 described [1] . These vectors can be transformed, such as Agrobacterium tumefaciens. Then this transformed Agrobacterium treated plant cells and transformed so plant cells regenerate into whole drousha protein according to the present invention (or combinations of such proteins), it is also possible encoding other proteins can be introduced into plant cells by other known methods, including the use of microsurgey gun, electroporation, electrotransformation, microinjection and so on, and that the regeneration of transformed plant cells is carried out according to methods known in the art, including treatment of cells with cytokines, if it is necessary or desirable in order to increase the frequency of regeneration.

In addition, appropriate organisms (e.g. micro-organisms, for which the production of proteins of the present invention is not significantly toxic) can be transformed by a vector containing a gene (or genes) encoding a protein, such that the transformed microorganisms produce this protein. Microorganisms can contain, in addition, genes encoding other proteins, such as PIN protein and/or protein, the sequence of which is described in one or more of the PEFC. N 4-6. In addition, these additional proteins may also contain various chitinases and/or glucanase. The most preferred such protein is the chitinase 4, as described [1].

Such microorganisms can be sayti dried and spray on infected plants or plants with the threat of infection.

The list of sequences is given at the end of the description.

LITERATURE

1. PCT Patent Application N. PCT/DK92/00108, Publication N. WO 92/17591.

2. Hejgaard et al., FEBS Letters, 307, 389-392 (1992).

1. The protein having fungicidal activity and having an amino acid sequence selected from the group of amino acid sequences SEQ ID NO : 1, SEQ ID NO : 2 and SEQ ID NO : 3.

2. Recombinant DNA encoding a protein having fungicidal activity under item 1.

3. Recombinant DNA under item 2, having the sequence represented in SEQ ID NO 7.

4. A method of combating fungi, which are exposed to the protein in p. 1.

 

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