RU2522833C2 - Method of obtaining nanostructured material based on recombinant archea flagella - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology and can be used for obtaining a nanostructured material based on recombinant flagella of archea H. salinarum, bound with metal ions or nanoparticles. Transformed with a recombinant plasmid cells of archea are grown, flagella, containing peptide inserts for binding with metal ions or nanoparticles, are separated. The surface of flagella is modified by binding peptide inserts with the said ions or nanoparticles with further washing, drying and packaging of the obtained material. The plasmid construction contains recombinant genes for synthesis of A1 and A2 flagella-forming flagellins. A sequence of flagellin A1 and/or flagellin A2 contains a peptide insert for selective binding metal ions or nanoparticles, where the location of the peptide insert is determined in the region between the first and second sites of glycosylation, located in flagellin A1 between position 86 and position 96 SEQ ID NO: 2, and in flagllin A2 between position 82 and position 92 SEQ ID NO: 3.

EFFECT: invention makes it possible to obtain the nanostructured material with improved adhesive properties, resistant with respect to impact of high temperatures.

10 cl, 7 dwg, 7 ex

Description

Technical field

The invention relates to the field of biotechnology, genetic and protein engineering, to nanotechnological applications using archaea flagella and can be used in the construction of diagnostic devices, battery cells, in medicine and in the pharmaceutical industry.

State of the art

Currently, great efforts are aimed at finding new nanomaterials that can be useful in developing a wide range of devices, the activity of which depends on the contact area of the active zones located on the surface of the devices.

Such devices include sensory sensors [1], components for creating elements of electronics, batteries, displays [2], coating of biological and medical devices requiring the absence of aggressive chemical components [3], such components can also include coating or composition components in the composition of pharmacological preparations [4].

Known inventions in which biological coating materials based on peptides [4] or viruses [2, 5-9] are used as coating elements. In order to form a developed surface structure, viruses are used, on the surface of which both particles of inorganic substances and metal ions are immobilized [5-10]. In the practice of creating surfaces with a developed surface structure, special attention is paid to options that include protein structures, including amino acids that can non-covalently bind metal ions [5-16].

In order for the nanostructured coating to be held on the surface of the developed devices, adhesive compounds are used [3]. However, the introduction of adhesive compounds is not always possible, especially in biomedical applications. Therefore, it is most optimal to use materials that themselves have adhesive properties. The choice of biological materials for creating nanostructures with a developed surface, ensuring the operation and preservation of biologically active zones, is not an easy task.

Known inventions in which to create developed surfaces using nanomaterials made on the basis of the M13 virus [5-10]. However, viral structures do not retain their properties in solutions with high temperature [5-10].

It is known that the flagella of archaea retain their structure at high temperatures [17]. In addition, it is known that materials made on the basis of flagella have sufficient adhesive properties and can be used to create molecular adhesives. In the invention [3], issues of obtaining adhesive materials containing at least one protein obtained from archaeal flagellins are considered. The application shows that archaeal flagellins adhere well not only to organic materials, such as the cell wall, but also to surfaces made from inorganic materials such as metals, plastics, and quartz. The invention indicates that not all flagellins have this possibility, so for H. Salinarum, only FlaB1 and FlaB2 are listed as flagellins in the application, which are not the main proteins of the flagellum, but play the connecting role between the cellular motor and the long spiral filament of the flagellum, consisting of A-flagellins [17]. The reason for this interest is scientific information on the adhesive role of some flagellins in the flagellum and the subsequent search for homologous flagellin sequences among all archaea with known genomic sequences. As the main material of molecular glue, the authors use individual flagellin proteins, from which the glue material is formed. The application for the invention pays great attention to methods for isolating target flagellins from flagella, which implies the irreversible separation of flagella into components and the destruction of their structure.

However, in this invention [3], the mechanism of high adhesion of flagellins to a wide class of inorganic substances is not disclosed, and places on the surface of flagellins that play the role of molecular glue are not disclosed. At the same time, the authors believe that substitutions of up to 60 amino acids can be introduced in the structures of the flagellins under consideration, and the place and composition of substitutions and insertions are not indicated for any type of flagellin. To clarify the possibility of such substitutions, additional studies of the properties of flagellins obtained by genetic engineering method are required. The specified invention does not confirm the possibility and effectiveness of the use of genetically engineered flagellins for effective non-covalent bonding of metal ions on its surface.

The aim of the present invention is to expand the types of coatings with a developed surface structure based on nanomaterials, on the basis of which it is possible to provide non-covalent bonding of metal ions, metal nanoparticles, semiconductors, with the aim of forming the surface of sensors, battery plates, non-woven materials with new properties.

Another objective of the invention is to obtain a technical result that prevents the influence of high temperatures on the safety of the surface structure of the nanomaterial and its adhesive properties.

The next objective of the invention is to increase the binding efficiency of metal ions, metal nanoparticles, semiconductors compared with materials obtained from wild-type archaea.

These technical results are ensured by the use of recombinant flagellin flagella as nanomaterials, wherein at least one peptide insert is introduced into at least one flagellin protein, the amino acid composition of which allows an effective non-covalent bond with the corresponding at least one type of metal ion, metal nanoparticles, semiconductor.

SUMMARY OF THE INVENTION

Objects of the invention are recombinant proteins of flagellin A1 and flagellin A2, used to form different types of flagella of the archaea Halobacterium salinarum with increased activity of binding of metal ions, metal nanoparticles, semiconductors, compared with their wild-type counterparts. To increase the binding activity of metal ions, metal nanoparticles, semiconductors, at least one peptide insert in the region between the first and second glycosylation sites is included in the wild-type flagellin sequence A1 (SEQ ID NO: 2) and A2 (SEQ ID NO: 3) . These regions include the sequence SEQ ID NO: 6 of flagellin A1 and the sequence of SEQ ID NO: 7 of flagellin A2.

Other objects of the invention are peptide insert sequences, which may contain a single sequence of 6 to 60 amino acids in length, or the insert contains short sequentially linked sequences with a total insert length of up to 60 amino acids, with the number of short sequences ranging from 2 to 10 .

The sequence of the peptide insert is chosen so that it non-covalently binds metal ions selected from the group consisting of: cobalt, vanadium, nickel, manganese, iron, cadmium, tungsten, chromium, zirconium, titanium, scandium, yttrium, copper, calcium, aluminum, barium, beryllium, magnesium and strontium, and / or metal nanoparticles selected from the group consisting of gold, platinum, silver, palladium, or nanoparticles having semiconductor properties: for example ZnS, CdS, as well as nanoparticles having magnetic properties, eg FePt, CoPt.

The following objects of the invention are plasmid DNA based on pNXA plasmid for expression of different types of recombinant flagella of the archaea N. Salinarum with increased efficiency of binding of metal ions, metal nanoparticles, semiconductors, compared with their wild-type analogues. The composition of the plasmids includes H. Salinarum A-flagellin operon, which contains successively placed regions A1 and A2 of flagellin genes at the XbaI and HindIII cloning sites, the ampicillin resistance gene AmpR, the Novobiocin resistance gene NovR, and the halophilic b-galactosidase bgaH gene. Depending on the type of recombinant flagellum, plasmids encode: a) a recombinant flagellin A1 protein with a peptide insert and a recombinant protein encoding wild-type flagellin A2 (SEQ ID NO: 3), b) a recombinant flagellin A2 protein with a peptide insert and a recombinant protein, wild-type encoding flagellin A1 (SEQ ID NO: 2), c) a recombinant flagellin A1 protein with a peptide insert and a recombinant flagellin A2 protein with a peptide insert.

The following variants of objects of the invention are strains based on H. Salinarum R1 as producers of different types of recombinant archaea flagella containing antibiotic resistance: cells obtained by transforming H. Salinarum R1 cells with recombinant pNX DNA plasmids encoding a) a recombinant flagellin A1 protein with a peptide insert and a recombinant protein encoding wild-type flagellin A2 (SEQ ID NO: 3), b) a recombinant flagellin A2 protein with a peptide insert and a recombinant protein encoding wild-type flagellin A1 (SEQ ID NO: 2 ), c) a recombinant flagellin A1 protein with a peptide insert and a recombinant flagellin A2 protein with a peptide insert.

The following variants of objects of the invention are methods for producing nanostructured material based on recombinant archaea flagella. These methods provide the following steps: a) synthesis of recombinant archaea flagella by culturing a producer strain, b) isolation of archaea flagella containing peptide inserts for non-covalent binding, c) surface modification of flagella by binding of peptide inserts to metal ions, metal or semiconductor nanoparticles, g ) oxidation of metal ions; e) washing, drying and packaging of the obtained nanostructured material.

The following variants of objects of the invention are compositions of a nanostructured material based on archaea flagella, which are made in the form of a solution or lyophilized form and include at least one type of recombinant flagellum, consisting of a) a recombinant flagellin A1 protein with a peptide insert and a recombinant a protein encoding wild-type flagellin A2 (SEQ ID NO: 3), b) a recombinant flagellin A2 protein with a peptide insert and a recombinant protein encoding wild-type flagellin A1 (SEQ ID NO: 2), c) recombinant flagellin A1 protein with a peptide insert; and recombinant flagellin A2 protein with a peptide insert. At the same time, the surfaces of the recombinant flagella are modified and the compositions additionally contain acceptable additives for forming the coating material. Moreover, the composition is made in the form of a solution or lyophilized form and includes acceptable additives for forming the coating material.

The following variants of objects of the invention are coating materials for forming a nanostructured coating of plates, films, capsules, textile material containing at least one composition comprising at least one type of modified recombinant flagellum and distributed at least in part integumentary material.

The following variants of objects of the invention are kits for forming articles from archaea flagella, containing at least a container, a composition of recombinant archaea flagella and packaging material.

List of figures

Figure 1 presents the physical map of the plasmid pNXA.

Figure 2 presents the amino acid sequence of A1-flagellin. The sequences of glycosylation sites are highlighted.

Figure 3 presents the amino acid sequence of A2-flagellin. The sequences of glycosylation sites are highlighted.

Figure 4 shows the nucleotide and its corresponding amino acid sequence of A1-flagellin in the region of the inserts, as well as the sequence of the inserts themselves. The sequences of the glycosylation sites and the sequence of the BstXI restriction site are in bold.

Figure 5 shows the nucleotide and its corresponding amino acid sequence of A2-flagellin in the region of the inserts after the introduction of the AluNHI restriction endonuclease site, as well as the sequence of the inserts themselves. The sequences of the glycosylation sites and the sequence of the AluNHI restriction site are in bold.

6. An electron microscopic image of the flagella H. salinarum is presented: (A) wild type, (B) modified flagella with an insert in the A1-flagellin FLAG peptide; and H. salinarum cells: (B) wild type, (D) FLAG strain. The preparations were labeled with specific antibodies and protein A conjugates with colloidal gold particles (10 nm). Negative contrast with uranyl acetate. The scale bar is 300 nm.

7. An electron microscopic image of N. salinarum flagella is presented: (A) wild type, (B-C) modified flagella with an insert of the FLAG peptide in A1-flagellin. Flagella were mineralized with cobalt oxide (A, B) and copper oxide (C). Negative contrast was performed with uranyl acetate. The scale bar is 300 nm.

Description

A study of the properties of extended N. Salinarum flagella (12 nm in diameter and several micrometers long) based on wild-type flagellins A1 and A2 (without genetically engineered inserts) showed that flagellin flagella have adhesive properties on the surface of metals, plastics, and other materials . However, such flagella have low binding efficiency of metal ions on their surface, which does not allow the use of wild-type flagellin to create nanomaterials with desired properties. To obtain a nanostructured material with desired properties, the polymer structure of the flagellum, consisting of A1 and A2 flagellins, it is necessary to give the property to specifically bind metal ions or nanoparticles. To accomplish such a task, it is necessary to select regions suitable for inclusion of peptide inserts in the flagellin sequences, with the possibility of their subsequent modification by metal ions. In this case, the structural mechanisms ensuring the polymerization of flagellins A1 and A2 into the flagellum must be preserved, and for the effective modification of the inserts, it is necessary to ensure the peptide inserts are removed to the external surface of the formed flagellum. The result is a combined nanostructured fiber material containing filaments of modified flagella.

It is known that flagellins A1 and A2, which form the composition of the flagella H.salinarum N, are glycosylated, with each flagellin containing up to 3 glycosylation sites (NXT, NXS sequences) [17-18].

In the process of studying the properties of flagella, it was found that the introduction of short peptide inserts in the sites located between the glycosylation sites of flagellins does not affect the formation of the structure of the flagella, and the inserts themselves during the synthesis of flagella remain exposed on the outer surface of the flagella, which makes it possible for efficient non-covalent binding of amino acids that are part of the inserts with ions and nanoparticles of metaolls.

In the general case, the method for producing recombinant archaea flagella consists of the following stages?

A) Isolation of DNA encoding flagelin.

B) The choice of the type of plasmid and its genetic design.

B) Selection of a sequence encoding an insert peptide.

D) The choice of the place in the amino acid sequence of the internal flagellin fragment for the peptide insert, as well as the choice of restriction endonuclease to cut the sequence at the selected location.

E) Formation of a plasmid.

E) Cell selection and cell transformation with the obtained plasmid.

G) The cultivation of cells on the surface of an agar medium and the subsequent growth of cells in a liquid medium.

H) Isolation of flagella from the culture medium for modification and subsequent mineralization of the surface of the flagella.

I) Modification of the flagella by the selected metal ion / or surface modification by metal nanoparticles and subsequent mineralization of the flagellum surface by the selected substance.

K) Isolation, drying and storage of mineralized flagella intended for the formation of material.

Isolation of DNA encoding flagelin.

To isolate total DNA from H.salinarum, as well as to isolate plasmid DNA from E. coli, glass-filled columns from Qiagen (USA) are used. DNA is isolated according to the manufacturer's instructions. Total H.salinarum DNA is used as a template for generating PCR fragments of the A-flagellin operon with flanking regions.

The choice of the type of plasmid and its genetic design.

The recombinant pNXA plasmid DNA shown in FIG. 1 has a molecule size of about 11,000 bp. and contains the cloned A-flagellin operon H. Salinarum containing regions A1 and A2 of the flagellin genes at the XbaI and HindIII cloning sites. This plasmid was obtained on the basis of the pNX plasmid [19] by cloning the PCR fragment of H. Salinarum A-flagellin operon, and the length of the cloned fragment is about 2400 nucleotides (SEQ ID NO: 1). The plasmid contains the ampicillin resistance gene (AmpR) and the novobiocin resistance gene (NovR). The plasmid also contains the gene for halophilic b-galactosidase (bgaH). The AmpR gene and the bacterial replication initiation site allow this plasmid and its modifications to be expanded in E. coli cells. The NovR gene ensures the resistance of transformed archaea cells to novobiocin, the bgaH gene product allows the transformed cells to turn blue by the decomposition products of the commercial X-Gal substrate, which serves as an additional marker for confirming the transformation. The haloarchaeic site of replication initiation is absent in the plasmid. This leads to the fact that upon transformation with a plasmid, archaea cells acquire resistance to novobiocin only when the plasmid is inserted into the chromosome by the mechanism of homologous recombination. In the construction of the plasmid is about 1200 bp in sum, the flagellin A1 gene shown in FIG. 2 (SEQ ID NO: 2) and the flagellin A2 gene shown in FIG. 3 (SEQ ID NO: 3) as well as the left (about 600 bp) and the right (400 bp) flanking portions of the A-flagellin operon [26]. In addition, genes A1 and A2 are separated by an intergenic gap 11 nucleotides long. The DNA fragment of the A-flagellin operon was produced by primers:

5'-GTCGGTGCTCTAGACGTCGGGGATCGG-3 '(SEQ ID NO: 4)

5'-TCACGATGCAAAGCTTCCATCAGTACGG-3 '(SEQ ID NO: 5)

using PCR. The fragment was cloned into the plasmid at the HindIII and XbaI restriction sites. Depending on the variants of the genetic construction of the plasmid, a sequence encoding a peptide insert is introduced into the region of the A1 gene, or the A2 gene, or simultaneously in the region of A1 and A2 of the gene.

Selecting a region in the flagellin amino acid sequence for a peptide insert.

The introduction of peptide inserts is carried out in the field of A1 and A2 flagellins located between their first two glycosylation sites from NLT to NLS. For A1-flagellin, the sequence of the region looks like VRQAAGADNI (SEQ ID NO: 6), and for A2-flagellin, this sequence looks like VRQAAGADNI (SEQ ID NO: 7). It is permissible to insert at any place between these glycosylation sites, from pos. 86 to pos. 96 in the area of flagellin A1 (Figure 2) and between pos. 82 to pos. 92 in the area of flaggel A2 (Figure 3). It is preferable to insert the insert in the middle of the area between poses. 89 to pos. 92 for flagellin A1. So figure 2 presents the amino acid sequence of A1-flagellin, and the sequence of glycosylation sites are highlighted. For flagellin A2, it is preferable to introduce an insert in the region between poses. 85 to pos. 88. Figure 3 shows the amino acid sequence of A2-flagellin, and the sequence of glycosylation sites is also highlighted. In this case, the introduced amino acid sequence is exposed to the surface of the formed flagellum, which allows its subsequent processing with substances having an affinity for the used insert.

Cloning of sequences encoding peptide inserts Cloning of sequences encoding a selected peptide in the region of the A1 and / or A2 gene is carried out using standard techniques, including: 1) selection of a restriction site in the region of genes corresponding to the amino acid sequences of protein fragments (SEQ ID NO: 6) and (SEQ ID NO: 7), 2) the synthesis of paired oligonucleotides, one of which encodes a peptide insert, and the other is complementary to it to form a duplex. Also, these oligonucleotides must contain sticky ends for the selected restriction site, 3) Cloning of the duplex at the selected site. The sequence of the peptide insert contains a single sequence with a length of 6 to 60 amino acids or contains short sequentially connected to each other sequences with a total insert length of up to 60 amino acids, with the number of short sequences ranging from 2 to 10.

The following is an example of cloning sequences encoding a selected peptide. So, for example, for the A1 gene, such cloning consisted of the steps: 1) the BstXI restriction endonuclease was selected, which cuts the flagellin A1 gene at the site corresponding to 90-91 amino acid of the protein, 2) the oligonucleotide sequences SEQ ID NO: 21, SEQ were synthesized ID NO: 22, 3) cloned the oligonucleotide duplex into the pNXA plasmid (Figure 4). Moreover, all oligonucleotide sequences encoding the selected peptide can be schematically depicted as follows: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 3'-end for cloning at the BstXI restriction site: 5'-NN (NNN) xGCCG-3 '(where N is the standard designation of the nucleotide). The second oligonucleotide is complementary to the first for duplex formation and, in addition, contains a sticky end for cloning at the BstXI site: 5 '- (NNN) xNNCGGC-3' (where N is the standard nucleotide designation). A diagram of the method of introducing an insert into the A1 gene is shown in FIG. 4. The number x determines the number of codons encoding the amino acid and can reach values from 6 to 60 codons for a single insert. If it becomes necessary to introduce an insert from pos. 86 to pos. 96 in the area of flagellin A1 and between pos. 82 to pos. 92 in the area of flaggelin A2, but it is not possible to select a suitable site for restriction endonuclease, it is possible in this area to change the nucleotide and, accordingly, amino acid sequence. Such a replacement does not lead to loss of flagellin functionality and can, if necessary, be carried out both on the A1 and A2 flagellin genes, if it contains additional amino acid substitutions with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity, with any amino acid substitution being preferably conservative.

For the A2 gene, such cloning consisted of the following steps: 1) the nucleotide sequence of the A2 gene with the pNXA plasmid was replaced with synthetic primers SEQ ID NO: 23, SEQ ID NO: 24 using the Stratagen QuikChange site-directed mutagenesis kit ( since it was not possible to select a suitable restriction endonuclease). This substitution led to the appearance of the AsuNHI restriction site in the A2 gene, and in the corresponding amino acid sequence gene there was a substitution in pos. 86 of alanine for leucine SEQ ID NO: 8, 2) oligonucleotide sequences were synthesized, for example, SEQ ID NO: 25, SEQ ID NO : 26, 3) the duplex of oligonucleotides was cloned into the pNXA plasmid (Figure 5) In this case, all oligonucleotide sequences encoding the selected peptide can be schematically depicted as follows: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 5 ' -end for cloning at the AsuNHI restriction site: 5'-CTAGNN (NNN) x-3 '(where N is the standard nucleotide designation). The second oligonucleotide is complementary to the first one to form a duplex and, in addition, contains a sticky end for cloning at the AsuNHI site: 5'-CTAG (NNN) xNN-3 '(where N is the standard nucleotide designation). A diagram of a method for introducing an insert into an A2 gene is shown in FIG. 5. The number x determines the number of codons encoding the amino acid and can reach values from 6 to 60 codons for a single insert.

Obtaining plasmids with tandem-cloned inserts of the listed sequences and obtaining producer strains based on them may possibly enhance or improve individual characteristics of the resulting nanomaterial.

If the insert is repeated in tandem, then the value of x should decrease by a multiple of the number of repeating inserts.

The selection of the sequence encoding the peptide of the insert.

Peptide sequences are known that have the ability to bind certain ligands [5-13, 15, 16]. A strain of halophilic archaeon H. salinarum, synthesizing flagella modified with the FLAG peptide DYKDDDDK (SEQ ID NO: 9), was created as one of the examples confirming the reproducibility and operability of the method for producing a nanostructured material based on archaea flagella. This example (see example 1) includes, but is not limited to other variants of flagella modified with peptide sequences having high affinity for various ligands.

Similarly, archaea flagellin inserts modified by other surface exposed inserts can be obtained with preservation of the extended supramolecular structure of flagella. In the light of the available data, it seems advisable to obtain N. Salinarum flagella with modified A1 and / or A2 flagellin by the following variants of peptide sequences that bind charged ions, metal oxides and nanoparticles, iron phosphate, bind ligands with semiconductor and magnetic properties.

The tetraglutamine EEEE sequence (SEQ ID NO: 10) binds positively charged metal ions. Flagella modified by this sequence can be used to obtain material for electrodes of lithium-ion batteries [5-6]. In addition, it was shown that this tetrapeptide is able to bind iron phosphate, which is also a promising material for the cathodes of lithium-ion batteries [20].

For some coatings using nanostructured materials, archaea flagella can be used with peptide inserts that bind colloidal nanoparticles of noble metals, or archaea flagella containing two types of inserts, one of them binds metal ions, the other nanoparticles of noble metals. An example of an insert that binds gold nanoparticles to the surface of the flagellum is the so-called gold-binding peptide motif LKAHLPPSRLPS (SEQ ID NO: 11) [5-6], which includes, but is not limited to, the types of peptide inserts for binding nanoparticles on the surface of the archaic flagellum.

Perhaps the combined use of two different types of peptide inserts (SEQ ID NO: 11) and (SEQ ID NO: 10), which, for example, were jointly expressed to obtain bifunctional particles of the modified M13 virus [5-6].

As the peptide insert, peptide sequences can be used that can bind ligands that provide semiconductor and magnetic properties. So the peptide with the sequence CNNPMHQNC (SEQ ID NO: 12) binds ZnS, the peptide with the sequence SLTPLTTSHLRS (SEQ ID NO: 13) binds CdS, the peptide with the sequence HNKHLPSTQPLA (SEQ ID NO: 14) binds FePt, the peptide with the sequence CNAGDHANC (SEQ IDQ: QQ NO: 15) binds CoPt [10].

The tetrapeptides RRRR (SEQ ID NO: 16) and RKRK (SEQ ID NO: 17) are known to bind metal ions and oxides. Vapors have selective affinity for Cu2O and ZnO, respectively [21].

The peptide structures that form the loops have the ability to bind metal ions, and, depending on the composition, have anionic or cationic properties. Such loops include His-loop histidine loops (GHHHHHH) (SEQ ID NO: 18), Glu-Asp loop glutamine-asparagine loops (DQDQDQG) (SEQ ID NO: 19), Arg-lysine loops Arg- Lys loop "(RKRKRKR) (SEQ ID NO: 20) [11].

Plasmid variants

To verify the effectiveness of the invention, the following plasmid variants were obtained on the basis of pNXA plasmid: 1) recombinant plasmid DNA pNXA1FLAG, which provides synthesis of FLAG-modified A1-flagellin and unmodified A2-flagellin, 2) recombinant plasmid DNA pNXA1A2FLAG, which provides synthesis of modified A-FLNAG p1XA1F2AG - and A2-flagellins, 3) recombinant plasmid DNA pNXA1FLAGA2GBP, which provides the synthesis of modified FLAG-peptide A1-flagellin and modified gold-binding peptide A2-flagellin in N. Sal cells inarum R1 during the transformation and insertion of this plasmid into the chromosome of the body. Options for plasmids and methods for their preparation are shown in example 2.

All of the listed plasmid DNAs were obtained by single insertion of each sequence at the indicated sites. However, these sequences can also be inserted in tandem 2 or more times, which will lead to an extension of the inserted encoded peptide by a multiple of its length and can lead to the synthesis of flagellins, which differ in properties not only from the native, but also from the listed modifications of flagellins.

Cell selection and cell transformation with the obtained plasmid

The obtained plasmids transform H. salinarum cells and grow them on the surface of an agar medium with an antibiotic. The grown cell colonies are subcultured into a liquid medium, and after growth, either flagella are isolated from the cells or small numbers of cells are selected.

To obtain producer strains of modified flagellins, N. Salinarum cells are transformed according to the described method [22]. H. salinarum is an extreme halophilic gram-negative archaeon with obligate aerobicity. The natural habitat of this organism is the water of the Dead Sea, and the extremely high salinity (up to 5.5 M NaCl) is optimal for growth. Under laboratory conditions, it is grown on a medium of the following composition: 25% NaCl, 0.2% KCl, 2% Mg2SO4 7 × H2O, 0.3% Na citrate, 0.5% tryptone, 0.2% yeast extract, pH 7.5 at 37 ° C [23].

H. salinarum is a single rod-shaped cell that does not form spores, having a lofotrichial arrangement of flagella; amphitrichial localization is possible in the stationary phase in N. salinarum cells. In all cases, the filaments of the flagella form a bundle, the rotation of which creates a hydrodynamic force that sets the cell in motion. The bundle includes 5-10 threads, which are a semi-rigid spiral. With the growth of H. salinarum, a significant amount of free flagella is found in the culture medium.

Suspensions of cells in liquid nutrient media, or colonies grown on the surface of an agarized medium, are red due to the presence of bactorodopsin in the cell wall.

In Russia, the strain H. salinarum R1 is available, provided by the All-Russian Collection of Industrial Microorganisms (Moscow). All obtained producer strains based on H. Salinarum R1 are characterized by the following features:

- morphological signs: rod-shaped cells, gram-negative, non-spore,

- cultural characteristics: when growing on an agar medium for N. Salinarum, the colonies are round, smooth, shiny, the edge is even, painted red. When growing on liquid media, they form an intense smooth haze,

- physical and biological signs: cells grow at a temperature of from 20 ° C to 45 ° C with an optimum pH of 6.8 to 7.5. As a source of nitrogen, both mineral salts in the ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. As a carbon source, amino acids, glycerin, carbohydrates,

- resistance to antibiotics: cells show resistance to novobiocin (up to 0.6 μg / ml), due to the presence of the NovR gene in the pNXA plasmid,

- producer strains differ from the recipient strain H. Salinarum R1 only in the presence of various variants of the recombinant plasmid DNA pNXA, which gives it resistance to novobiocin,

- the absence of a haloarchean site for the initiation of replication leads to the fact that when a plasmid is transformed, resistance to novobiocin is only acquired by archaea cells when the plasmid is inserted into the chromosome by the mechanism of homologous recombination,

- producer strains, in addition to the modified A-flagellin operon integrated with the plasmid, also contain the native A-operon. Therefore, in addition to modified flagellins (its presence was shown experimentally by immuno-electron microscopy), flagella may also contain unmodified flagellins.

The following steps for producing recombinant flagella, such as a) growing cells on the surface of an agar medium and subsequent cell growth in a liquid medium, b) isolating flagella from the culture medium for modification and subsequent mineralization of the flagellum surface, d) modifying the flagella with a selected metal ion and / or modification surface with metal or semiconductor nanoparticles and then subsequent mineralization of the surface of the flagella with the selected substance, e) isolation, drying and storage of mineralized flagella, intended chennyh for forming the material - are given in Examples 4-6.

Of the examples of modified flagella, flagella with an insert of a FLAG peptide (SEQ ID NO: 9) allow detecting its presence on the surface using specific monoclonal antibodies. Using immuno-electron microscopy of both the isolated preparation of flagella and fixed cells of the FLAG strain of H. salinarum, it was shown that this peptide is present in flagellin, and the flagella are specifically labeled with antibodies. It is seen that the modified flagella retain their native helical structure and do not morphologically differ from wild-type flagella, while the FLAG peptide is available for binding to antibodies, i.e., it is part of the surface region of the A1-flagellin polypetid chain (Fig. 6). It is known that tetraglutamine or tetraasparaginic sequences brought to the surface of proteins are able to efficiently bind metal ions (for example, Co2 +, Cu2 +) [5-13]. Since the FLAG peptide also contains 4 consecutive negatively charged amino acid residues, aspartate, the flagella were tested for binding of metal ions. The loop formed by eight residues was sufficient to bring a cluster of charged amino acids to the surface of the flagellum and able to bind metal ions. Then, ions in the presence of sodium borohydride were transferred to insoluble oxide particles attached to the flagellum (Fig. 7). The resulting structures resemble nanostructured materials obtained by other researchers based on viral particles and bacterial flagella. It is believed that such materials, depending on the type of bound ligand, can be used as a material for electrodes of lithium-ion batteries, various nanosensors and catalysts, and the production of materials with magnetic and semiconductor properties [3-16].

Examples

Example 1. Obtaining genetic constructs

Genetic constructs are obtained on the basis of plasmid pNX [19], which contains the ampicillin resistance gene and the novobiocin resistance gene. To isolate total DNA from H.salinarum, as well as to isolate plasmid DNA from E. coli, glass-filled columns from Qiagen (USA) are used. DNA is isolated according to the manufacturer's instructions. The total DNA of H. salinarum is used as a matrix for the production of PCR fragments of the A-flagellin operon with flanking regions.

Using oligonucleotide primers SEQ ID NO: 4 and SEQ ID NO: 5, PCR is carried out in 50 μl of a mixture consisting of PCR buffer (for Taq DNA polymerase: 57 mM Tris-HCl, pH 8.8, 16.6 mM (NH) 2SO4, 0.1 % Tween-20) with a MgCl2 concentration of 1.5 to 2.5 mM, a mixture of deoxyribonucleotides (at a concentration of 0.2 mM each), primer oligonucleotides of 20-100 pM each, 2-20 ng plasmid or 100-200 ng chromosomal DNA, and a mixture of 1 -2 units Taq DNA polymerase and 0.1-0.2 units. Pfu DNA polymerase. The duration of the stages of melting, annealing, and elongation is 10 s, 30 s, and 3 min, respectively. The annealing temperature of the primers is determined by the maximum yield of products, the melting stage is carried out at 95 ° C, and elongation at 72 ° C, the number of cycles is 25.

PCR products as well as plasmid DNA were analyzed by agarose gel electrophoresis in TAE buffer according to [24]. DNA processing with restriction endonucleoses (all companies of Fermentas, Lithuania) and ligation using T4 DNA ligase (Fermentas, Lithuania) are carried out according to the manufacturer's instructions.

Example 2. Design plasmids with different types of peptide inserts

Various duplexes of synthetic oligonucleotides were used as a cloned sequence: (SEQ ID NO: 21), (SEQ ID NO: 22); (SEQ ID NO: 25). (SEQ ID NO: 26); (SEQ ID NO: 27), (SEQ ID NO: 28). All sequences in accordance with complementarity are annealed in pairs on top of each other.

A) Construction of plasmid pNXA1FLAG

As an example, which includes, but is not limited to other options, this example discusses a method of introducing an insert that encodes the sequence of the FLAG peptide. For this purpose, a primer with the sequence of SEQ ID NO: 21 and a primer with the complementary sequence of SEQ ID NO: 22 are used.

The primers are annealed on top of each other with cooling of their mixture from 80 ° C to room temperature, after which they are phosphorylated at the 3'-ends by polynucleotide kinase (Fermentas, Lithuania) according to the instructions. Next, cloning of this synthetic double-stranded oligonucleotide sequence encoding a FLAG peptide is carried out by inserting flagellin into the A1 gene at the site corresponding to 90-91 amino acid of the protein at the BstXI restriction site, due to the sticky ends for this site (Figure 4), formed at annealing of synthetic single chain oligonucleotides (SEQ ID NO: 21) and (SEQ ID NO: 22). The obtained ligase mixture was used to transform E. coli cells according to [25] and plated on plates with agarized (1.5%) LB medium with 100 μg / ml ampicillin. To produce plasmid DNA, the grown colony from a cup is subcultured into a liquid medium with an antibiotic, and a plasmid is isolated after the cells undergrowth. The resulting plasmids are sequenced on an ABI PRIZM 310 DNA sequencer (Applied Biosystems, USA).

Thus, in the above example, to clone oligonucleotides encoding other inserts at the BstXI restriction site in A1-geneflagellin, they should look like this: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 3'-end: 5'-NN (NNN) xGCCG-3 '(where N is the standard designation of the nucleotide). The second oligonucleotide is complementary to the first for duplex formation and, in addition, contains a sticky end for cloning at the BstXI site: 5 '- (NNN) xNNCGGC-3' (where N is the standard nucleotide designation). A diagram of the method of introducing an insert into the A1 gene is shown in FIG. 4. The number x determines the number of codons encoding the amino acid and can reach values from 6 to 60 codons for a single insert.

B) Construction of plasmid pNXA1A2FLAG

Plasmid pNXA1A2FLAG was obtained in two stages: firstly, conditions were created for the insertion of different sequences into the region of the A2 flagellin gene. Since there is no restriction site for BstXI in the A2 gene, and it was also not possible to select other unique sites in the selected region between the glycosylation sites, it was decided to introduce an artificial site by replacing several nucleotides. This replacement was carried out in the plasmid pNXA using synthetic oligunucleotides

5'-CACCGTGCGCCAGCTAGCCGGAGCC-3 '(SEQ ID NO: 23) and

5'-GGCTCCGGCTAGCTGGCGCACGGTG-3 '(SEQ ID NO: 24)

and the QuikChange site-directed mutagenesis kit (Stratagene). Thus, the site for the restriction endonuclease AluNHI was introduced, leading to the replacement in the product of A2 gene 86 of the amino acid alanine with leucine. As a result, the flagellin A2 fragment bounded by the first two glycosylation sites, after replacement, looks like VRQLAGADNI (SEQ ID NO: 8). Such a replacement does not lead to the loss of flagellin functionality and can, if necessary, be carried out both on the A1- and A2-genes of flagellins. The number of substitutions in the region of 10 amino acids between the first two glycosylation sites in both A1 and A2 flagellins can reach 20% percent while retaining at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity, with any amino acid substitution being preferably a conservative substitution. Next, a synthetic double-stranded oligonucleotide sequence encoding a FLAG peptide was inserted into the A1 gene at the site corresponding to 90-91 amino acids and the A2 gene at the site corresponding to 86-87 amino acids of each protein. This sequence is inserted into the A1 flagellin gene at the BstXI restriction site due to the sticky ends for this site resulting from the annealing of synthetic single-chain oligonucleotides (SEQ ID NO: 21) and (SEQ ID NO: 22), and into the A2 flagellin gene at the site AluNHI restriction also due to the sticky ends for a given site resulting from the annealing of synthetic single-stranded oligonucleotides (Figures 4-5).

5'-CTAGGTGACTACAAGGACGATGACGATAAGGGT-3 '(SEQ ID NO: 25)

5'-CTAGACCCTTATCGTCATCGTCCTTGTAGTCAC-3 '(SEQ ID NO: 26)

Thus, in the above example, to clone oligonucleotides encoding other inserts at the BstXI restriction site in A1-geneflagellin, they should look like this: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 3'-end: 5'-NN (NNN ) xGCCG-3 '(where N is the standard designation of the nucleotide). The second oligonucleotide is complementary to the first for duplex formation and, in addition, contains a sticky end for cloning at the BstXI site: 5 '- (NNN) xNNCGGC-3' (where N is the standard nucleotide designation). A diagram of the method of introducing an insert into the A1 gene is shown in FIG. 4. For insertion into the A2 gene: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 5'-end for cloning at the AsuNHI restriction site: 5'-CTAGNN (NNN) x-3 '(where N is the standard nucleotide designation). The second oligonucleotide is complementary to the first one to form a duplex and, in addition, contains a sticky end for cloning at the AsuNHI site: 5'-CTAG (NNN) xNN-3 '(where N is the standard nucleotide designation). A diagram of a method for introducing an insert into an A2 gene is shown in FIG. 5. The number x determines the number of codons encoding the amino acid, and can reach values from 6 to 60 codons for a single insert.

B) Construction of plasmid pNXA1FLAGA2GBP

Plasmid pNXA1FLAGA2GBP was also obtained in two stages: first, similarly to the preparation of plasmid pNXA1A2FLAG, a restriction endonuclease AluNHI site was introduced into the region of the A2 flagellin gene, which leads to the replacement of the alanine amino acid 86 with leucine in the A2 gene product. Next, a synthetic double-stranded oligonucleotide sequence encoding a FLAG peptide was inserted into the A1 gene at the site corresponding to 90-91 amino acid of the protein, and the synthetic double-stranded oligonucleotide sequence (SEQ ID NO) into the A2 gene at the site corresponding to 86-87 amino acid of the protein : 11) encoding a peptide that binds colloidal gold nanoparticles (Golden Binding Peptide). The sequence of the gold-binding peptide motif (SEQ ID NO: 11) was determined by phage display. The sequence encoding the FLAG peptide is inserted into the A1 flagellin gene at the BstXI restriction site due to the sticky ends for this site resulting from the annealing of two synthetic single-stranded oligonucleotides (SEQ ID NO: 21) and (SEQ ID NO: 22). The sequence (SEQ ID NO: 11) encoding the peptide was inserted into the A2-flagellin gene at the AluNHI restriction site also due to the sticky ends for this site resulting from the annealing of synthetic single-stranded oligonucleotides (Figs. 4-5):

5'-CTAGGTCTAAAGGCCCATCTACCTCCTAGTAGGCTACCTAGTGGT-3 '

(SEQ ID NO: 27)

5'-CAGATTTCCGGGTAGATGGAGGATCATCCGATGGATCACCAGATC-3 '

(SEQ ID NO: 28).

Thus, in the above example, to clone oligonucleotides encoding other inserts at the BstXI restriction site into the A1 flagellin gene, they should look like this: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 3'-end: 5'-NN ( NNN) xGCCG-3 '(where N is the standard designation of the nucleotide). The second oligonucleotide is complementary to the first for duplex formation and, in addition, contains a sticky end for cloning at the BstXI site: 5 '- (NNN) xNNCGGC-3' (where N is the standard nucleotide designation). A diagram of the method of introducing an insert into the A1 gene is shown in FIG. 4. For insertion into the A2 gene: the first oligonucleotide encodes the sequence of the inserted peptide and also contains a sticky 5'-end for cloning at the AsuNHI restriction site: 5'-CTAGNN (NNN) x-3 (where N is the standard nucleotide designation). The second oligonucleotide is complementary to the first one to form a duplex and, in addition, contains a sticky end for cloning at the AsuNHI site: 5'-CTAG (NNN) xNN-3 '(where N is the standard nucleotide designation). A diagram of a method for introducing an insert into an A2 gene is shown in FIG. 5. The number x determines the number of codons encoding the amino acid, and can reach values from 6 to 60 codons for a single insert.

Example 3. Obtaining strains of producers of modified flagella N. salinarum R1

Used strain N. salinarum R1, provided by the All-Russian collection of industrial microorganisms (Moscow). N. salinarum cells are grown on a medium of the following composition: 25% NaCl, 0.2% KCl, 2% Mg2S04 7x H20. 0.3% Na citrate, 0.5% tryptone, 0.2% yeast extract, pH 7.5 at 37 ° С [23].

The strain is obtained by transforming H. salinarum cells using polyethylene glycol (PEG) according to the previously described method [22] with the following modifications. Spheroplasts are obtained separately for each aliquot (1.5 ml) of the cell culture. Cells are pelleted in 1.5 ml tubes by centrifugation at 4000 g for 10 min and then resuspended in 150 μl spheroplasting solution. To obtain spheroplasts, 15 μl of 0.5 M EDTA was added to the cell suspension and the mixture was kept for 10 min at room temperature. Then add 10 μl of a solution containing about 5 μg of plasmid DNA, and the mixture is incubated for 5 minutes, after which an equal volume of the solution containing 60% PEG-600 and 40% spheroplasting solution (weight / volume) is added. Cells are incubated for 20 min at room temperature. To restore cells, 15 ml of culture medium containing 15% sucrose are added to the suspension and incubated for 12-24 hours at 37 ° С with shaking. The selection of transformants is carried out on an agar medium containing 15% sucrose and 0.1-0.2 μg / ml of the antibiotic novobiocin (Sigma, USA). Incubation is carried out at 37 ° C for 10-14 days. Cell cultures of H. salinarum are stored in Petri dishes on appropriate media with 1.5% agar at room temperature.

The obtained strains are cultured similarly to the untransformed N. Salinarum R1 cells with the exception of the addition of 0.1-0.2 μg / ml novobiocin antibiotic

Example 4. Isolation and purification of flagella

To isolate the flagella, the H. salinarum culture was grown in liter flasks from 200 ml of medium to the late stationary phase. The resulting biomass is precipitated by centrifugation at 8000 g for 30 minutes. To the supernatant obtained after cell precipitation, 4% PEG-6000 is added and then the flagella are precipitated by centrifugation at 15,000 g for 45 minutes. Further, CsCl density gradient centrifugation was used to clean the flagella (12 hours at 55,000 rpm, VTI-80 rotor, Beckman, USA). Cesium chloride is dissolved in saline to obtain a density of 1.36 g / cm ³ . The fraction containing flagella formed a visible zone in the middle of the tube. The contents of the fraction are diluted (10 times) with brine containing 25% NaCl, 20 mM MgSO4, and then the flagella are reprecipitated by centrifugation at 80,000 g for 1 hour.

Example 5. The binding of metal ions and the mineralization of flagella.

The binding of metal ions is carried out similarly to the method described by Belcher [5-6] with modifications. Flagella at a concentration of 0.1 mg / ml were incubated for one hour in an aqueous solution of 1.5 M NaCl containing 5 mM Co or Cu chloride. Then, one volume of a solution containing 1.5 M NaCl and 100 mM NaBH4 was added to three volumes of a flagella solution and incubated for 1 hour until the reaction stopped.

Example 6. Obtaining a nanostructured material based on mineralized flagella with modifications.

After mineralization of the modified flagella (example 4), they turn into an easily precipitated (4000g, 10 minutes) black precipitate (color of Co and Cu oxides). This precipitate is separated from the supernatant, then suspended in water and re-precipitated for washing from salt. The resulting precipitate is transferred to an inert substrate, dried under vacuum at room temperature for a day. The resulting dry black powder is stored at room temperature for a long time.

Example 7. Obtaining electron micrographs and the study of flagella using immuno-electron microscopy

To obtain electron micrographs, a JEM-100 microscope with JEOL (Japan) was used. To prepare electron microscopic samples, the preparations are applied to electron microscopic copper grids with a form-film substrate, stand for 1 min and the solution is taken out with filter paper, then the grid is placed on a solution of 2% uranyl acetate, incubated for 30 seconds, the solution is taken out with filter paper and then dried .

Samples of flagella are immediately applied to electron microscopic copper grids with a formar film substrate and incubated for 5 minutes. To prepare IEM preparations, H. salinarum cells are pre-fixed. For this, the cells are precipitated at 4000 g for 5 minutes and then suspended in a 15% NaCl solution, to which glutaraldehyde is added to a concentration of 1%, and left for 20 minutes. Then the cell suspension was placed on electron microscopic copper grids with a formar film substrate and incubated for 5 min, after which the grids were placed on a solution of 0.7% glycine in 5% NaCl to bind the excess aldehyde. Grids with samples of flagella or fixed cells are placed on the IEM blocking buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1% BSA) for 30 min, and then transferred to the same buffer with the preparation of monoclonal ANTI FLAG M2 antibodies (Sigma, USA) and incubated for 30 minutes Antibodies are bred 500, 200 and 100 times. Then the nets are washed from antibodies in the blocking IEM buffer 3-4 times for 1 min and transferred to the same buffer with addition of protein A conjugates with colloidal gold particles (particle size 10 nm, Jannsen Biotech, Belgium) for 45-60 minutes. Conjugates are diluted 50, 20 and 10 times. Next, the mesh is washed with a blocking buffer for IEM without BSA 2-3 times for 5-10 minutes, the solution is removed with filter paper and placed on a solution of 2% uranyl acetate for negative contrast. After 30 s, a solution of 2% uranyl acetate was removed with filter paper, the grids were dried, and samples were examined under a microscope. The best labeling of flagella preparations was achieved by dilution of antibodies 200-100, and conjugates 20-10 times.

Although the present invention describes specific and preferred options for its implementation, it should be borne in mind that these options do not limit the possible modifications and variations of the present invention.

Literature

[1] Bock L. et al. Nano-Chem-Fet Based Biosensors. US Pat. Applic. No. 20080044911 (February 21, 2008).

[2] Iran B.Q. NANO-electronics. US Pat. Applic. No. 20070285843 (December 13, 2007).

[3] Wirth R. et al. Molecular Glue. US Pat. Applic. No. 200880305524 (December 11, 2008).

[4] Gazit E. et al. Peptide nanostructures encapsulating a foreign material and method of manufacturing same. US Pat. Applic. No. 200,000,7455 (April 13, 2006).

[5] Nam K.T. et al. Virus-Enabled Synthesis and Assembly of Nanowires for Lithium Ion Battery Electrodes. Science, V.312, P.885-888 (2006).

[6] Belcher A.M. et al. Nanoscaling ordering of hybrid materials using genetically engineered mesoscale virus. US Pat. Applic. No. 30030073104 (April 17, 2003).

[7] Belcher A.M. et al. Multifunctional biomaterials as scaffolds for electronic, optical, magnetic, semiconducting, and biotechnological applications. US Pat. Applic. No. 20050170336 (August 4, 2005).

[8] Belcher A.M. et al. Biological control ofnanoparticle nucleation, shape and crystal phase US Pat. Applic. No. 20060275791 (December 7, 2006).

[9] Nam K.T. et al. Virus scaffold for self-assembled, flexible and light lithium battery. US Pat. Applic. No. 20060121346 (June 8, 2006).

[10] Mao S. et al., Virus-Based Toolkit for the Directed Synthesis of Magnetic and Semiconducting Nanowires. Science, 2004, V.303, P.213-217.

[11] Kumara M.T. et al., Self-assembly of metal nanoparticles and nanotubes on bioengineered flagella scaffolds. Chem. Mater., 2007, V.19, P.2056-2064.

[12] Yu B. et al., A Novel Biometallic Interface: High Affinity Tip-Associated Binding by Pilin-Derived Protein Nanotubes. J. Bionanosci., 2007, V.1, 73-83.

[13] Audette G.F., Hazes B. Development of protein nanotubes from a multi-purpose biological structure. J. Nanosci. Nanotechnol., 2007, V.7, P.2222-2229.

[14] Atabekov I.G. The use of viral structures as tools for nanotechnology. Ross Nanotech., 2008, T.3, R.132-141.

[15] Mudalige Thilak Kumara et al., Bioengineered Flagella Protein Nanotubes with Cysteine Loops: Self-Assembly and Manipulation in an Optical Trap Nano Lett., Vol.6, No.9, 2006.

[16] Benita Westerlund-Wikstro et al., Functional expression of adhesive peptides as fusions to Escherichia coli flagellin. Protein Engineering vol. 10 no.11 pp. 1319-1326, 1997.

[17] Thomas A.N. et al., The archaeal flagellum: a different kind of prokaryotic motility structure. FEMS Microbiol. Reviews, 2001, V.25, P.147-174.

[18]] Logan S.M. Flagellar glycosylation - a new component of the motility repertoire? Microbiol., 2006, V.152, P.1249-1262.

[19] Tarasov V.Y., Pyatibratov M.G., Tang S., Dyall-Smith M., Fedorov O.V. // Role of flagellins from A and B loci in flagella formation of Halobacterium salinarum. Mol. Microbiol., 2000, V.35, P.69-78.

[20] Fabricating Genetically Engineered High-Power Lithium Ion Batteries Using Multiple Virus Genes. Yun Jung Lee, Hyunjung Yi, Woo-Jae Kirn, Kisuk Kang, Dong Soo Yun, Michael S. Strano, Gerbrand Ceder, Angela M. Belcher. April 2, 2009 / 10.1126 / science.

[21] Corrine K. Thai et al., Identification and Characterization of Cu2O- and ZnO-Binding Polypeptides by Escherichia coli Cell Surface Display: Toward an Understanding of Metal Oxide Binding. BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 2, JULY 20, 2004.

[22] Cline S.W. et al., Transformation methods for halophilic archaebacteria. Can. J. Microbiol., 1989, V.35, P.148-152.

[23] Dyall-Smith M. The halohandbook: protocols for halobacterial genetics. 2008 (http://www.haloarchaea.com/resources/halohandbook).

[24] Sambrook J., Russell D.W. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.

[25] Inoue H. et al., High efficiency transformation of Escherichia coli with plasmids. Gene, 1990, V.96, P.23-28.

[26] Oesterhelt The system was conceived and implemented within the Dept. of Membrane Biochemistry of the Max-Planck-Institute of Biochemistry, Martinsried, Germany (http://www.halolex.mpg.de/public)/

Claims (10)

1. A method of obtaining a nanostructured material based on recombinant archaea of N. archinum salinarum flagella associated with metal ions or nanoparticles, comprising transforming archaea cells with a recombinant plasmid, growing cells, isolation of archaea flagella containing peptide inserts for binding with metal ions or nanoparticles or with nanoparticles with magnetic or semiconductor properties, modifying the surface of flagella by binding peptide inserts to metal, semiconductor, magnetic nanoparticles or metal ions, with the possibility of subsequent mineralization of these ions or nanoparticles and washing, drying and packaging of the obtained nanostructured material, where the plasmid design contains recombinant genes for the synthesis of flagellins A1 and A2, forming a flagellum, while the sequence of flagellin A1 and / or flagellin A2 contains a peptide insert for selective binding of metal ions or nanoparticles, where the location of the peptide insert is determined in the region between the first and second glycosylation sites, located in flagellin A1 between position 86 and position 96 of SEQ ID NO: 2, and in flagellin A2 between position 82 and position 92 of SEQ ID NO: 3, where the peptide insert may contain a single sequence of 6 to 60 amino acids in length or contain short sequentially sequences connected to each other with a total insertion length of up to 60 amino acids, with the number of short sequences ranging from 2 to 10. 2. The method according to claim 1, characterized in that the genetic design of the recombinant plasmid is obtained on the basis of plasmid pNXA. 3. The method according to claim 1, characterized in that the surface modification of the flagella is carried out by binding peptide inserts with metal ions, followed by mineralization. 4. The method according to claim 3, characterized in that the sequence of the peptide insert is chosen so that it non-covalently binds metal ions selected from the group consisting of cobalt, vanadium, nickel, manganese, iron, cadmium, tungsten, chromium, zirconium, titanium, scandium, yttrium, copper, calcium, aluminum, barium, beryllium, magnesium and strontium. 5. The method according to claim 1, characterized in that the surface modification of the flagella is carried out by binding peptide inserts with metal, semiconductor, magnetic nanoparticles. 6. The method according to claim 5, characterized in that the sequence of the peptide insert is chosen so that it non-covalently binds nanoparticles of metals selected from the group consisting of gold, platinum, silver, palladium. 7. The method according to claim 5, characterized in that the sequence of the peptide insert is chosen so that it non-covalently binds nanoparticles having semiconductor properties belonging to the group: ZnS, CdS. 8. The method according to claim 5, characterized in that the sequence of the peptide insert is chosen so that it non-covalently binds nanoparticles having magnetic properties included in the group: FePt, CoPt. 9. The method according to claim 1, characterized in that the peptide insert is selected from the group consisting of amino acid sequences of SEQ ID NO: 9-20. 10. The method according to claim 9, characterized in that the peptide insert is selected from the group consisting of amino acid sequences of SEQ ID NO: 9 and 11.

Images