The selected polypeptide selected protein (options) and isolated protein (options) to obtain nanostructures

 

(57) Abstract:

The invention relates to structures of nanometer size used to construct the microscopic and macroscopic structures. The selected polypeptide is a variant gp36 protein of bacteriophage T4. Amino acid sequence is shown in the description. Selected protein consists of the first part of gp37 protein of T-even-like bacteriophage and the last 10 - 60 C-terminal amino acids gp36 protein or the first part of the gp36 protein and the second part of the protein gp34 T-even-like bacteriophage. Amino acid sequences listed in the description. Isolated protein comprises 20 contiguous amino acids gp37 protein, or gp36 protein, or protein gp34 T-even-like bacteriophage. Amino acid sequences listed in the description. The invention allows to create materials whose properties can be tailored to the specific requirements of technology nanometer scale, to improve the strength and consistency of macrostructure. 6 C. p. F.-ly, 9 Il.

The invention relates to nanostructures, i.e., structures of nanometer size used to construct the microscopic and macroscopic structures. Mainly, this options.

Although in accordance with theoretical assumptions of the strength of most materials based on metals and ceramics is a consequence of the strength of the bonds between molecules of their components and the surfaces of the crystallites, such strength is substantially limited by the defects in their crystalline and glassy structures. Such defects are typically caused by the original materials or arise during manufacture and are often spread due to the effects of environmental loads.

Developing field of nanotechnology has increased restrictions on traditional materials. The possibility of designing and production of very small structures (i.e. structures of nanometer size), able to perform complex functions, depends on the use of appropriate materials, which can be affected in a predictable and reproducible methods that possess the properties required for each new application.

Biological systems are examples of complex nanostructures. Living cells produce proteins and combine them into patterns, which are formed perfectly and are resistant to damage in their normal environment. In some lucentio polypeptides. Finally, proteins are subjected to the processes of correction, providing a high degree of quality control.

In connection with the above, in the art there is a need for methods and compositions using such unique features of proteins for the formation of the constituent parts of synthetic nanostructures. This need is to create materials whose properties can be tailored to the specific requirements of technology nanometer scale. In addition, because the subunits of most macro-structural materials, ceramics, metals, fibers, etc. based on nanostructure binding subunits, the manufacturer of the respective subunits without defects, with accurate dimensions and homogeneity, should improve the strength and consistency of macrostructure as surfaces become more organized and can interact more tightly on extensive areas than larger heterogeneous material.

In accordance with one aspect of the present invention provides for isolated protein building blocks for nanostructures containing modified proteins of filamentous outgrowth bacteriophage what properties. Specific internal peptide sequences can be subjected to deletion without affecting their ability to form dimers and be associated with their natural partners filiform process. On the other hand, they can be modified so that they will only communicate with other modified and not native partners filiform process; to be thermolabile interactions with their partners; or contain additional functional groups, allowing their interaction with heterologous binding fragments.

The present invention also covers the slit proteins that contain sequences of two or more different proteins of filamentous outgrowth. Protein gp35, which forms the gusset modify so that the formed average angles other than the natural average angle 137o(7oor 156o( 12o), and was carried out thermolabile interactions with its partners.

In accordance with another aspect of the present invention provides nanostructures containing the native and modified proteins of filamentous outgrowth of bacteriophage T4. Such nitya layers, or open three-dimensional camera, or closed solid objects.

In Fig. 1A and 1B presents a schematic of the particles of the bacteriophage T4 (Fig. 1A) and a schematic representation of the thread ridge of the bacteriophage T4 (Fig. 1B).

In Fig. 2 presents a schematic representation of the elementary block.

Fig. 3A-3D depict schematic view: one-dimensional iterative rod connected along the x-axis (Fig. 3A); closed simple layers (Fig. 3B); closed layers of brick masonry (Fig. 3C); and open layers in the form of masonry (Fig. 3D).

In Fig. 4 shows a schematic illustration of the two basic units used to construct porous and solid layers (top and bottom), which, when alternately applying form a stacked set of cameras shown in the drawing.

Fig. 5 depicts a schematic view of the corner structure with an angle of 120o.

Fig. 6 depicts the DNA sequence (SEQ 1A N:1) gene 34, 35, 36 and 37 of the bacteriophage T4.

Fig. 7 depicts the amino acid sequence (represented by one-letter codes), the gene products of genes 34 (SEQ 1D N:2, ORFX SEQ 1D N:3), 35 (SEQ 1D N:4), 36 (SEQ 1D N:5) and 37 (SEQ 1D N:6) of bacteriophage T4. Amino acid A (upper line of each pair). It should be noted that the deduced protein sequence of the gene 35 (from the database NCB1), apparently, cannot be regarded as precise.

In Fig. 8A-8B presents a schematic picture: education P37 dimeric initiator of molecules transformed by self-Assembly in the dimer (Fig. 8A); and education P37 trimeric initiator molecule, turned in a result of self-Assembly in the trimer (Fig. 8B).

In Fig. 9 presents a schematic representation of polymer formation (P37-36)nwith the help of the initiator, which represents a dimer formed during self-Assembly.

A detailed description of the invention.

All patents, patent applications and literature references cited in the description, are fully covered by the references contained in this document. In the event of any inconsistency preference, including definitions, the present description.

Although the present invention is described with reference mainly to proteins of filamentous outgrowth of bacteriophage T4, it should be borne in mind that the invention is also applicable to proteins of filamentous outgrowth of another phage, similar to the T-even phage, for example, a family of T4 (for example, T4, Tu1a, Th1b) and family T2 (T2, T6, K3, Ox2, M1, etc).

The term "Chimera" in the text are indicated chimeric proteins in which at least the amino - and carboxy-terminal region are derived from different source polypeptides regardless of whether the original polypeptides polypeptides naturally occurring or modified as a result of mutagenesis.

Used in the text the term "homodimer" refers to the sets of almost identical protein subunits, forming a pronounced three-dimensional structure.

The designation "gp" refers to the Monomeric polypeptide, whereas the symbol "P" refers to homologoumena. P34, P36, and P37, presumably, represent homodimer or homotrimer.

The reference in the text of the isolated polypeptide, which mainly consists of a specific amino acid sequence, refers to a polypeptide with a specific sequence or the polypeptide comprising conservative substitutions in this sequence. As should be clear to the person skilled in the art, conservative substitutions are substitutions in which the acid residue substituted for a residue. The scope of the invention also covers a polypeptide, which lacks one or more amino acids at the amino end or carboxy-late, up to five at either end, when the absence of specific residues has no significant impact on the structure or function of the polypeptide in the practical implementation of the present invention.

The present invention relates to a new class of protein building blocks whose dimensions are measured in nanometers, which are used in the construction of microscopic and macroscopic structures. Not limited to a particular theory, it is assumed that the main link is a glycosilated, consisting of two identical protein subunits, having a cross--conformation, although it is also possible three-dimensional structures. Thus, it should be clear that references to "glycosilated" or "dimerization" used in the text, in many cases, can be considered as referring also to homotrimer or trimerization. Such a long, hard and stable rod-like units can be combined with other terminals using cross-linking of devices that can join genetically or in vitro. The ends of a single rod can pricoediniacia and their characteristics flexibility makes possible the self-Assembly of various structures in situ. Thus, the unit cell can undergo self-Assembly in a predetermined larger structures with one, two or three dimensions. Self-Assembly can be carried out with the formation of structures with accurate dimensions and uniform strength in the defect-free biological production components. The rods can also be modified by genetic and chemical methods with the formation of certain specific areas of the accession of other chemical entities that provides for the formation of complex structures.

An important aspect of the present invention is the fact that the protein chains may be constructed so that they include rods of different lengths and may optionally be modified so that they include distinctive features that modify their surface properties in a certain way and/or affects their ability to connect with other identical or different links. In addition, the possibility of self-Assembly can be extended by obtaining chimeric proteins, combining the properties of two different representatives of this class. Such structural hallmark is achieved by Mani the actions and methods of the present invention uses the properties of natural proteins, i.e., the resulting patterns are hard, durable, resistant to water, heat, protease-resistant and can be biodegradable. A large number of links can be easily created in the micro-organisms. In addition, to facilitate automation of large quantities of parts and subaggregates can be stored and used as needed.

The sequence of the protein subunits based on the components threadlike outgrowth of the bacteriophage T4 E. coli. It should be understood that the principles and methods can be applied to the filamentary appendages other T-even phages or other related bacteriophages with similar Otrokovice and/or filamentous structures.

The structure of filamentous outgrowth of bacteriophage T4 (illustrated in Fig. 1) can schematically be represented as follows (N =amino-end, C= carboxy-end): N[P34]C - N[gp35]C - N[P36]C - N[P37]C. P34, P36, and P37 are rigid, rod-like protein homodimer, in which two identical folds oriented in the same direction, merge in the orientation of the surface - to surface as a result of hydrophobic interactions between superimposed on each other by seams and P37 are homotrimer). In contrast, gp35 is a Monomeric polypeptide that is specifically attached to the N-end P36 and then to the C-end of P34 and forms a corner connection between two terminals. In the course of infection of E. coli by bacteriophage T4 two gp37 monomer timeresults education P37 of glycosilated; it is assumed that the process of dimerization is initiated near the C-Terminus P37 and requires two proteins chaperones E. coli. (Variant gp37 with temperature-sensitive mutation near the C-end used in the present invention, requires the dimerization of the presence of only one chaperone, gp57). During the dimerization of the N-end P37 initiates dimerization of two P36 monomers P36 pin. The connection between the C-end of P36 and N-end P37 is dense and hard, but non-covalent. Then N-end P36 joins gp35 the monomer; this interaction stabilizes the P36 and forms a knee filiform process. Finally, gp35 is attached to the C-end of P34 (which uses gp57 for dimerization). Thus, the self-Assembly of filamentous outgrowth is regulated by specific order of the interaction of specific subunits, resulting in structural maturation caused by the formation of the first sub-aggregate, provides interaction with the random mixture components.

In accordance with the present invention, genes encoding such proteins, may be modified so that the rods are formed of different lengths with different combinations of endings. In this respect, the properties of native proteins have special advantages. First, the fold consists of antiparallel - chain s-curves on the left (L) and right (R) sides. Secondly, the amino acid side chains alternate and extend downward from the plane of the layer. The first property ensures elongation curves with the formation of symmetric and specific sites connection between L and R surfaces, and the formation of sites attach to other structures. In addition, the rod sections-folds can be shortened or lengthened as a result of genetic manipulation, for example by splicing the DNA regions encoding the bends on the same edge of the reservoir, with the formation of new curves, excluding the presence of interfering peptides, or by insertion of segments of the peptide is carried out similarly by splicing on the bend. The second property allows the amino acid chain to be extended above and below the surface folds modified by the method of genetic substitution or chemical crosslinking. Vagina. As should be clear to the person skilled in the art, such properties provide greater flexibility when designing units that can be collected with the formation of a large number of different organizations, some of which are discussed in detail below.

Structural units.

The rods of the present invention functions similar to the functions of a wooden 2 x 4 studs or steel bars used for construction. In this case, the surface accurately reproducible at the molecular level and therefore suitable for specific connections to the same or different terminals of the cells on fixed parts of the connection. Such surfaces also modify so that they were more or less hydrophilic, including positively or negatively charged groups, and had the tabs that are built for specific binding with other cells or with the intermediate connection of the two receptor sites. The surface of the rod and the schema element of the rod illustrated in Fig. 2. Three dimensions of the rod are indicated as follows: x, measured from the back side (B) to the front (F); Y, the dimension from the bottom (D) up (U); and Z, the dimension of the left (L) elemental rods, United along the x-axis (Fig. 3A), however, regular connection subjacent in any of the other two dimensions will also form an elongated structure, but with different cross-sections on the structure by the dimension x.

Two-dimensional structures represent the layers formed by the interaction of the rods along any two axes.

1) simple Closed layers are formed from surfaces that exactly overlap along any two axes (Fig. 3B). 2) Closed type layers of brickwork are formed as a result of interaction between the units having exactly the overlapping surface in one dimension and a special type of overlap in the other dimension (Fig. 3C). In this case, there may exist two different number of additional connections located at a distance of exactly 1/2 of the unit cell between them. If they are centered (i.e., each row of 1/4 from the end, every connection will appear in the centre of the cells located at the top and bottom. If they are shifted, then the connection will also be retracted. In this design advanced interactive sites schematically indicated as.and... If each of the interacting sites is symmetrical, it cheimaditida only interacting with rows, located in the same or opposite directions, get a layer of unidirectional cores or layers of rods in alternating directions.

3) Open the seams of the type of masonry (or grid) are formed when cells are separated from each other by a distance more than half of the cells (Fig. 3D). The size of the holes (or pores) depend on the distance (dx) separating the interacting sites and distance (dy), for which the sites are separated one surface from another.

Three-dimensional designs require the use of sterically compatible interactions between all three surfaces with the formation of solids. 1) Closed solid system can be collected from cells that exactly overlap in all three dimensions (for example, in the case of exact overlap of closed simple layers). Similarly, the closed reservoir type masonry can form a closed solid system in the exact overlapping layers or as a result of moving with the transfer of the brickwork in the third dimension. This option requires an appropriate set of connections in all three pairs of parallel outer surfaces of the cell. 2) a Porous solid system and cell exactly overlap in the third dimension, a solid system with many holes the exact dimensions perpendicular to the plane of the sheet. If instead you want a material with closed spatial elements with layers of width dz (i.e. in the dimension U--->D), then a simple closed layer is applied a layer to open the layer type of brickwork with the aim of closing holes. If the overlap of the open seam type masonry is, for example, 1/4 of the cell, to obtain a layer use the rod 3/4 length cell. Then the required connections in dimension Z. Two such cells are used for polymerization of such alternating layers, and the layers themselves schematically depicted in Fig. 4.

All of the above patterns consist of simple linear rods. The second cell corner cell, expanding the type and spatial configuration of possible structures. Corner cell connects two rods at an angle other than 180olike angle iron. The average angle and the degree of rigidity built in such a connecting structure. For example, the structure shown in Fig. 5, has an angle of 120oand various specific sites connection positions a and b. The following are examples of structures obtained with ispolzovanie, perpendicular to the direction of the web by adding angles, perpendicular to the seam. In this case a three-dimensional mesh structure. Attach the corners of the magnitude of the 90oto the ends of the rods forms an angle lying almost in the plane of the layer, which allows you to add new terminals to such corners (which should have some clearance outside the plane of the source layer for joining in the first place) with the formation of a new layer, almost parallel to the first orientation, normal with respect to its upper or lower neighbor.

2) Hexagons prepared from a mixture of rods and angular ligaments, forming angles of 120o. In this case, there are two exceptional set of connections. Each kit collected from one of two ends of the rod and one of the two complementary sites at an angle. The resulting structure is linear in the sense that the hexagon has a direction (either clockwise or counterclockwise). It can be turned into a two-dimensional open mesh (i.e. a two-dimensional honeycomb) by connecting the sides of the hexagon. It can form a hexagonal tube in the connection of the top of the bottom hexagon with the bottom facial surface verneau hexagonal tube.

3) Spiral hexagonal tubes get like hexagons, but the sixth cell is not connected with the first closing of the hexagon. Instead, the end of the move from the plane of the hexagon and add the seventh and following cells with the formation of the hexagonal tube, which may be a spring, if between cells of the spiral are little or no strength of adhesion, or they can be a hard rod, if such a power exists in the immediate vicinity of the joined cells.

The specialist in this area should be clear that the compositions and methods of the present invention also encompass other polygonal patterns, such as the octagon, solid and open systems, such as the tetrahedron and dvadtsatoshnika formed from Treugolnik, and cubes obtained from squares and rectangles. The range of structures is limited only by the types of angular cells and substituents, which may be constructed along different axes of rod cells. For example, other naturally occurring angles found in the threads of bacteriophage T7 with the 90o(With Steven al., J. Mol.Biol. 200:352-365, 1988).

Design and obtaining core deposits, based on the four polypeptides, which are part of the filiform outgrowths of bacteriophage T4, i.e., gp34, gp35, gp36 and gp37. Genes encoding such proteins have been cloned and have been installed in their DNA and protein sequences (genes 36 and 37, see Oliver with TCS. , J. Mol. Biol. 153: 545-568, 1981). DNA and amino acid sequences of genes 34, 35, 36 and 37 are presented in the following Fig. 6 and 7.

Gp34, gp35, gp36 and gp37 received in a natural way after infection of E. coli cells with intact particles T4 phage. After synthesis in the cytoplasm of bacterial cells gp34, 36 and 37 of the monomers forming homodimeric sufficient for Assembly in ripening phage particles. Thus, E. coli serves as an effective and convenient factory for synthesis and dimerization protein subjacent described below.

In the practical implementation of the present invention, genes encoding proteins considered (natural, modified or recombinant), introducing the vectors in the expression of DNA, which are well known in this field. Such circular plasmids usually contain breeding marker genes (usually giving antibiotic resistant transformed bacteria), sequence, allowing replicable relatively inducible promoter, and also the site of binding to the ribosome. Examples of commercially available vectors that are suitable for use in the present invention, can serve as the pET system (Novagen, Inc., Madison, W1) and the vectors Superliner pSE380 and pSE280 (Invitrogen, CA San Diego, CA).

Used strategy is to 1) constructing a gene of interest and its cloning into the multiple cloning site; 2) transformation of E. coli cells with the recombinant plasmid; 3) inducing the expression of the cloned gene; 4) testing for the synthesis of the protein product; and finally, 5) testing for the formation of functional homodimers. In some cases, additional genes also clone in the same plasmid, when their function is required for dimerization protein of interest. For example, when expression of gp37 wild-type or versions connect bacterial chironomy gene 57; when the expression of gp36 wild-type or modified gp36 connect version of wild-type or a modified version of gp37 gene. Modified gp37 must have the ability to dimerization and contain N-end, which may be accompanied by dimerization of gp36. This method allows the formation of Monomeric gene products and, in nacatoch proteins, usually present in T4-infected cells.

Stage 1-4 described above strategies are implemented using methods well known in the field of recombinant DNA technology and protein expression in bacteria. For example, in stage 1 the splitting of restrictase in multiple sites with subsequent legirovaniem fragments used to construct deletions in the inner rod segment gp34, 36 and 37 (see below, Example 1). On the other hand, the splitting of a single or multiple restrictase followed ectonucleoside digesting (EXO - SIZE, New England, Biolabs, Beverly, MA) are used for deletion of DNA sequences in one or both directions from the original site of cleavage; when combined with the subsequent stage ligating this method provides education nesting series of deletions with increasing size. Similarly, the standard methods used for the reconstruction of segments of DNA from two different genes of filamentous process with the formation of chimeric genes encoding the fused protein (referred to in this description of "chimeras"). Generally, the latter method is used to provide alternating N - and C-ends, and, thus, to create new to the measures of this type, merge gp37-36 presented and described in Example 2. The preferred hosts for the production of such proteins (stage 2) are E. coli strain BL21 (DE3) and BL21 (DE3/pLysS) (obtained commercially from Novagen, Madison, Wl), although you can also use other compatible recA strains, such as HMS 174 (D3) and HMS 174 (DE3/pLysS). Transformation with recombinant plasmid (stage 2) is performed by standard methods (J. Sambrook, Molecular Cloning, Cold Spring Harbor Laboratories, Cold Spring Harbor, N, V; this book is also the source of standard methods using recombinant DNA used in the present invention). Transformed bacteria are selected based on their resistance to antibiotics, such as ampicillin and kanamycin. The method, which is induced by the expression of cloned genes filiform process (stage 3), depends on the nature of the promoter. The preferred promoter is the plac (Iaciq on the vector to reduce background expression), which can be adjusted by adding isopropylthioxanthone (IPTG). The second preferred promoter is a pT7/10, which is specific to T7 RNA polymerase and is not recognized by E. coli RNA polymerase. T7 RNA polymerase, is and BL21 (DE3) superrepressor Iaciq gene in the plasmid and induced and regulated by IPTG.

Typically a culture of the transformed bacteria are incubated in the presence of the inductor within a few hours, while monitoring the synthesis of a protein of interest. In this case, prepare extracts of bacterial cells and proteins of filamentous outgrowth T4 detects, for example, by the method of gel electrophoresis in the system sodium dodecyl sulphate (SDS) - polyacrylamide.

After detection of the modified protein in the bacterial extracts need to figure out forms whether it is appropriate homodimer (stage 4). Initially, this stage is carried out by testing for detection of this protein by anticorodal specific to Mature dimenisonal form of the protein.

Anticigarette specific to the tail fiber, prepared in accordance with the known method (Edgar R. S. u Lielausis I., Genetics 52:1187, 1965; Ward with TCS. J. Mol.Biol. 54:15, 1970). Briefly, this method consists in the fact that a T4 phage is used as immunogen; optional, the resulting anticigarette next adsorb attrastcive fagbemi particles, removing, thus, all antibodies except those that are directed against proteins of filamentous outgrowth. At a later stage, different aliquots of antisera ka. For example, if the extract contains only the components filiform process P34, gp35 and gp36 (produced from cells infected with mutant T4, which has no functional gp37 gene) is used for adsorption, then the resulting anticavity will recognize only Mature P37 and dimenisonal P36-P37. A similar approach can be used to prepare individual antisera that recognize only the Mature (i.e., homodimerization) P34 and P36 by adsorption extracts containing the distal half of the thread ridge or P34, gp35 and P37, respectively. An alternative may be the induction of antibodies against halves purified filamentous process, for example, P34 and gp35-P36-P37. Then anti-gp35-P36-P37 can adsorb P36-P37 obtaining anti-gp35, and anti-P36 can be obtained by adsorption using P37 and gp35. Anti-P37, anti-gp35 and anti-P34 can also be directly obtained using purified P37, gp35 and P34 as immunogens. Another approach is to develop specific monoclonal antibodies to different tail fiber components threadlike outgrowth or their segments.

Specific antibodies to sabyasachi or part of the process used any of the following with the global colonies subjected to screening in order to identify those systems, which Express the Mature proteins of filamentous outgrowth by direct transfer of the colonies, or, selectively, samples are lysed or deletirovannykh crops on nitrocellulose filters, implement, if necessary, lysis of bacterial cells on the filter and incubation in the presence of specific antibodies. The formation of immune complexes is then detected by methods commonly used in this field (for example, using secondary antibodies conjugated with chromogenic enzyme or radiolucency staphylococcal protein A). This method is particularly useful for screening large numbers of colonies, for example, those that are produced by the method EXO - SIZE deletions, as described above). 2) Bacterial cells expressing the protein, initially metabolic mark35S-methionine, and then prepare extracts and carry out the incubation in the presence of antisera. Then the immune complexes were recovered by incubation with immobilized protein A, followed by centrifugation, after which they can be separated using gel electrophoresis system SDS-polyacrylamide.

Alternative competitive analysis testing save internally deleteregvalue Belk is the Association with their respective partners, includes the use of in vitro complement system. 1) Bacterial extract containing interesting modified protein, as described above, is mixed with the second extract prepared from cells infected with T4 phage, representing a mutant in the gene of interest.

2) After several hours of incubation were added to the third extract, which contained a version of the wild-type to be checked protein, and incubation continued for several hours. 3) Finally, the extract was titrated for infectious phage particles by infection of E. coli and quantify the number of resulting sterile spots. The modified protein of filamentous outgrowth, right dimenisonal and able to connect with his partners included in the threads of the process of the dysfunctional way in stage 1, resulting prevented the inclusion of a version of the wild type protein in stage 2; the result of this operation was the reduction of the titer of the resulting phage sample. In contrast, if the modified protein is not able to timeresults and thus to form a proper N - and/or C-ends, it will not be included in the phage particles to a hundred, the engineers of the phage must be equal to the titer observed without the addition of the modified protein in stage 1 (negative control).

Another way to test the conservation of chimeras and internally deleteregvalue proteins of filamentous outgrowth ability to dimerization and Association with their respective partners is to conduct the experiment in vivo. This analysis allows to determine the ability of such chimeras and deletirovannykh proteins to compete with parts of normal phage for the Assembly, thereby reducing the size of the area of differentiation of wild-type phage infecting the same cell host, which is the recombinant expression of chimeras or subjected to deletions of proteins. Thus, expression of the expression vector encoding the Chimera or deleteriously protein, is induced within the cell, which then infect the wild-type phage. Inhibition of the production of wild-type phage demonstrates the ability of recombinant chimeras or protein Association with the corresponding proteins of filamentous outgrowth of this phage.

The above-described methods, as such or in combinations, are used in designing and obtaining various types of modified proteins nbor such which Express properly dimenisonal protein, allows the identification of positive colonies, which can then be individually tested by in vitro complementaly.

Examples of new proteins within the scope of the present invention without its limitations include:

1) Internally deleteregvalue gp34, 36 and 37 polypeptides (see below, example 1);

2) C-terminal truncated gp36, fused with the N-end N-terminal shortened gp37;

3) the Merger between gp36 and gp37, in which gp37 is an N-end relative to gp36 (i.e. the opposite of the natural order), denoted in the text by the term "gp37-36 Chimera" (see example 2);

4) the Merger between gp34 and gp36, in which gp36 represents the N-end to gp34 (i.e. in a direction opposite to the natural order), denoted in the text by the term "gp36-34 Chimera";

5) Option gp36, in which C is the end of mutated so that it loses the ability to interact (and response dimerization) N-end gp37 wild type designated in the text as "gp36*";

6) Option gp37, in which the N-terminal mutated in such a way that it forms P37, losing the ability to interact with the C-end of gp36 wild type designated in the text as "*

8) Option "P37-36-Chimera", which gp36 fragment is produced from the variant in accordance with 5), i.e. "P37-36*". (Relative clauses 5-8, see below, example 3).

9) Option "P37-36-Chimera", which gp37 fragment is produced from the variant according to 6) above, i.e. "*P37-36".

10) Option P37-36 chimeras,*P37-P36*in which fragments of gp36 and gp37 are made of the options in accordance with paragraph 7).

11) the Merger between gp36 and gp34, in which gp36 sequence placed N-terminal relative to gp34, dimer, which in the text are indicated as "P36-34 Chimera";

12) Options gp35, which form the average angles other than 137oor 158o/native angle/, for example, an angle less 125oor more 145oin conditions where a protein gp35 wild-type forms an angle 137owhen connecting to P34 and P36-P37-dimers, and/or manifests a greater or lesser flexibility than the native polypeptide;

13) Options gp34, 35, 36 and 37. which are thermolabile interactions or other variant-specific interaction with their cognate partners; and

14) Options gp37, in which the C-terminal domain of the polypeptide modify this Ober, the sequence produced from avidin that recognizes Biotin, consistency, produced from immunoglobulin heavy chain that recognizes A protein of Staphylococcus, consistency, produced from a Fab part of the heavy chain of monoclonal antibodies, which can join the appropriate copies of the light chain of the Fab and to form the binding site of the antigen, immunoactive sequence that recognizes specific antibodies, or sequences that bind specific metal ions. Such ligands can be immobilized to facilitate cleaning and/or Assembly.

In accordance with special technical solutions, chimeras of the invention contain part consisting of at least the first 10 (N-terminal) amino acids of the first protein of filamentous outgrowth, fused via peptide bond with a part consisting of at least the last 10 (C-terminal) amino acids of the second protein of filamentous outgrowth. The first and second proteins of filamentous outgrowth may be the same or different proteins. In accordance with another technical solution, chimeras contain amino acid part in the first 10-60 amino acids of the protein of filamentous outgrowth, merged with and is one technical solution, each amino acid is at least 20 amino acids of a protein of filamentous outgrowth. Such chimeras contain parts, i.e. polnorazmernyi proteins of filamentous outgrowth, fused with each other. In accordance with a preferred aspect of the first protein of filamentous process Chimera is taken from gp37, and the second protein of filamentous process - from gp36. This Chimera (gp37-36 Chimera), after oligomerization education P37-36 capable of polymerization in the other identical oligomers. gp36-34 Chimera, after oligomerization education P36-34, can communicate with the gp35, and such unit may further dry out.

In accordance with another technical solution, the first part is taken from gp37, and the second part of gp34. According to a preferred aspect, the chimeras of the invention is produced by insertions or deletions in the region of the turning structure of proteins of filamentous outgrowth. Most preferably, when inserted into the sequence of the filamentous process or merging with another sequence of the protein of filamentous outgrowth (preferably, in the manipulation on the level of recombinant DNA to give the desired encoded protein) is carried out in such a way that posledovatelnostei may also contain deletion constructs of proteins of filamentous outgrowth, which is shortened at one end, for example, does not have the amino - or carboxy-late (at least 5 or 10 amino acids) in the molecule. Such molecules, truncated at the amino-end, such as shortened gp37, gp34 or gp36, can be used as a "top" ("cap") nanostructures, because immediately after the introduction they tear up the polymerization. Such molecules preferably contain a fragment of the protein of filamentous outgrowth, which is missing at least the first 10, 20 or 60 aminoterminal amino acids.

To change the length of the core component of proteins, if desired, part of the same or different proteins of filamentous outgrowth can be inserted into the Chimera filiform process for lengthening the rod or be subjected to deletions of chimeras with the purpose of shortening rod.

Assembly of individual core components in nanostructures.

Expression of proteins of the present invention in E. coli, as described above, results in the synthesis of large quantities of protein and promotes the simultaneous expression and Assembly of the various components in the same cells. Ways to scale production of recombinant protein is simple and widely known in this field, and the th process of bacterial culture.

In accordance with the preferred technical solution of the native (non-recombinant) gp35 allocate for use by growing bacteriophage T4 with amber mutation in the gene 36, su0bacterial strain (non-amber-suppressor) and allocation gp35 from the resulting culture by standard methods.

P34, P36-P37, P37 and chimeras produced from them, purified from cultures of E. coli as a Mature dimers. gp35 and its variants purified as monomers. Purification is achieved using the following methods or combinations thereof using conventional methods: 1) chromatography on a molecular sieve, ionoobmennyh materials and/or hydrophobic matrices; 2) preparative ultracentrifugation; and (3) affinity chromatography using immobilized as a ligand-specific antibodies or other specific binding fragments. For example, C-terminal domain P37 associated with E. coli lipopolysaccharide Century, Other T4-like phages have P37 analogues that bind other components of the cell surface, such as OmpF or TSX protein. On the other hand, if proteins were genetically engineered so that they contain heterologous domains, the current ka is ISDE. For example, such heterologous domain can serve as Biotin, which binds with the streptavidin coated solid phase.

On the other hand, some components coexpressed in the same bacterial cells, and subassembly larger nanostructures are cleaned after the limited in vivo Assembly using the above methods.

Then purified components connected in vitro under such conditions, in which an Assembly of the desired nanostructures is carried out at temperatures in the range of from about 4oC to about 37oC and pH values in the range of from about 5 to about 9. For a given nanostructure optimal conditions assemblies (i.e., type and concentration of salts and metal ions) are easily determined by routine experimentation, for example, the change in each variable separately and monitoring of education of the respective products.

On the other hand can be prepared with one or more crude bacterial extracts were mixing and before cleaning carried out of the reaction Assembly.

In some cases, one or more purified components spontaneously assemble in the desired pattern without the use of initiators. In ists. This approach assumes the advantages of localization of the Assembly process (i.e., if the initiator immobilized or localize otherwise) and adjusting the size of the final structure. For example, the core components containing functional P36 C-end require functional P37 N-end to initiate the stoichiometric formation of the rod; therefore, the change in the relative amounts of initiator and core component will have an impact on the average length of the core polymer. If this ratio is equal to n, the average rod will have the following approximate structure (P37-36)n-N-the end P37-P37 C-end.

In other cases, the final nanostructure consists of two or more components that are not able to samsobeats separately, but only in combination with each other. In this situation, alternating cycles of Assembly can be

produce products of a precisely defined structure (see example 6B).

When using immobilized initiator may be desirable to remove the polymerized units from the matrix after the Assembly. For these purposes, by means of genetic engineering have created special is olabilirim (see the following example 5). In this way, the polymer can be easily separated from the associated matrix of the initiator, resulting in:

1) to easily obtain initial solutions of the homogeneous parts or subassembly and 2) reuse associated with the matrix initiator for the implementation of multiple cycles of initiation of polymerization, the growth of the polymer and is selected.

In accordance with the technical solution according to which the nanostructure is assembled so that it is attached to a solid matrix through gp34 /or P34/, one of the ways the Department nanostructures with its solution is the use of a mutant (thermolabile) gp34, which can be used to detach when exposed to high temperatures (for example, 40oC/. Such mutant gp34, called T4 tsB45 having a mutation in its C-end so that P34 is attached to the distal half filiform process 30oC, but can be separated from it in vitro as a result of incubation at 40oC in the presence of 1% SDS (unlike T4 wild type, which is stable in these conditions), was described previously (Seed, 1980, a study of the proximal half of the tail fiber of bacteriophage T4, abstract of thesis for the tx2">

Proteins that catalyze the formation of correct (with the lowest energy), sustainable secondary (2o) structure of proteins, called protein-chaperone. Often, especially in globular proteins, this stabilization is achieved by the tertiary structure, e.g., stabilization-folds as a result of their interaction in the cavities or as a result of interaction with a-helices. Usually chaperone prevent nutricare and interaction cross-links, which can lead to the formation of undesirable metastable folded intermediates and prevent or slow the appropriate styling. There are two additional protein, gp57 and gp38, in the morphogenesis of filiform appendages T4 phage, sometimes referred to as chaperone, because they are essential systems for the proper maturation of protein oligomers, but not in the final structures.

Normal system chaperones (for example, chaperonin/ES) interacts with some Oligopeptide fragments of the gene product to prevent undesirable interactions with Oligopeptide fragments in any other place of the same polypeptide or a different polypeptide. This can lead to the formation of a metastable is Noah (low-energy) state.

Gp57, probably together with some membrane proteins, plays a role in the imposition (and alignment) and/or the initiation of laying 2 or identical molecules gp37. Then the aligned peptides bind (zip) (mutually stabilizing their emerging-patterns) with the formation of the barrel without additional interaction with gp57. Gp57 performs functions in the Assembly of the T4 not only as a system that is involved in oligomerization gp37, but also gp34 and gp12.

Structural components for self-Assembly of trunks in vitro

On the other hand, to start the polymerization of chimeras using the previously derived chimeric or natural oligomeric unit, called the initiator, obtained in vivo, molecules (preferably peptides), capable of self-Assembly, can be obtained in the form of a fused protein, fused to the N-or C-end options filiform outgrowths of the present invention (chimeras, deletion/insertion structures to align their ends and facilitate their subsequent self-bending in oligomeric, sustainable folded rod-like (stem) cells in vitro, in the absence of the usually required for this process proteins chaperones (for example, gp57) and membrane be the and and polymerization. Usually the proper laying gp37 with P37 initiator requires infective phage cell membrane and two accompanying proteins chaperones gp38 and gp57. In accordance with a preferred technical solution, you can avoid having to use qp38 result from the application of mutation, ts3813 (duplication of 7 residues immediately in the forward direction from the transition zone gp37), which suppresses gene 38 (Wood, W. B., F. A. Eiserlinq, R. A. Crowther 1994, "Tait Long Fibers; Genes, Proteins, Structure, and Assembly" in Molecular Biology of Bacteriophage T4 (Jim D. Karam, Editor), American Society for Microbiology, Washington, D. C. p. 282-290). If a fragment of the self-assembling in the dimer or trimer or other oligomer ("self-assembling fragment) fused with C-terminal deletion gp37 in the forward or reverse direction from the transition region (transition region is a conserved region of 17 amino acid residues in T4-like proteins of filamentous process in which the protein structure is narrowed in a thin thread; see Henning et al., 1994, "Receptor recognition by T-even-type coliphages" in Molecular Biology of Bacteriophage T4, Karam (ed.), American Society for Microbiology, Washington, D. C., p. 291 - 298; Wood et al., 1994, "Long tail fibers; Genes, proteins, structure and assembly" in Molecular Biology of Bacteriophage T4, Karam (ed.), American Society for Microbiology, Washington, D. C., p. 282-290), then it is the expression of self-assembled fragment will oligomerizate poustie other proteins chaperones.

If P37 is a dimer /Fig. 8A/ self-assembling fragment can be sometimeswe peptide, such as lacinova "clasp", derived from residues 250-281 yeast transcription factor GCN4 (E. K. O'shea, R. Rutkowski u P. S. Kim, Science 243:538, 1989), or smodeliruite mutant Lazenby "fastened" peptide, p1L in which position "a" replaced by isoleucine, d - leucine (Harbury, P. C. , T. Zhang, P. S. Kim, T. Alper 1993. A. Switch Between Two-, Three-and Four-Stranded Coiled Coils in GC N 4 Leucine Zipper Mutants, Science, 262:1401-1407). If P37 is a trimer (Fig. 8B), the self-assembling fragment can be clotrimazole mutant Lazenby peptide type zipper, p11, in which position "a" and "d" replaced with isoleucine (Harbury, P. Century. al., ibid.). On the other hand, collagen peptide can be used as self-assembled fragment similar to that described with Bella al. (J. Bella, M. Baton, B. Brodsky, N. M., Berman. 1994. Crystal and Molecular Structure of Collagen-Like Peptide at Resolution, Science, 226:75-81), which samovyrivnyuyucha using insertional specific non-recurring alanine residue near the center.

Self-assembling fragments can be used to obtain the initiators of polymerization in the absence of normal ispolzovatsa gp37-36, gp37-36-C2as is illustrated in Fig. 9 (symbol C2means that the dimer-forming peptide fused to the C-end fragment gp36. This is used if the terminal is a dimeric structure. In another case, C3the trimer-forming peptide, fused to the C-end can be used for this operation). In addition, the use of N-end of the lac repressor of E. coli, for example, that is associated in the form of a tetramer, with the two helices pointing in each direction, can ensure that the two dimers or polymers of dimers) end to end, either at their N - or C-ends depending on what end are self-assembling peptides. They are also able to connect the N - and C-ends. In any case individually they are capable of forming only a dimer, each end which can be extended by adding the corresponding chimeric monomer (as shown for the simple case of Fig. 9).

In accordance with an alternative technical solution self-assembled fragment can be fused to the N-end of the chimeras. According to the specific technical solution of self-assembling fragment merges with at least a 10-amino acid part of the protein of filamentous outgrowth is Homer. For example, if the polymerization between the two beams is directed surface dimer-stitching, adding a heterodimeric unit, one surface of which is not promotiom further polymerization, can be very useful for closing the penultimate unit and result in breakage of polymerization. If two types of twisted areas of self-assembled fragment significantly more likely to be attracted to each other than themselves, all the dimers will be a hetero-dimers. This situation occurs for the N-terminal Jun and Fos latinovich areas type of zipper".

Another advantage of such heterodimeric units is the ability to carry out the polymerization and thus, over time, to create a single cell (or one surface in 2D row). For example, assume that the surface A is attached to B, but does not connect to itself ([A<->B] is used to denote a type of interaction). Let's mix A/A and B/B0(B0attached to the matrix for easier cleaning). Formed the B0/B - A/A. Now we shall wash A/A and add B/B. the resulting construction has the form B0/B-A/A-B/B. Add A/A0. The resulting design B

Application

The nanostructures of the present invention can be used in a variety of areas that require vysokopopulyarnyh, well-defined rows of filaments, cells, or solid systems, which may include specific areas attachment that allows linking to other materials.

In accordance with one of the technical solutions of three-dimensional hexagonal system of tubes is used as a molecular sieve or filter with regular vertical pores of a precise diameter, designed for selective separation of particles by size. Such filters can be used for sterilization of solutions (i.e., removal of microorganisms or viruses) or applied in the form of a series of filters with limited molecular-weight transmission. In this case, the protein components of the pores can be modified in such a way as to provide specific surface properties (i.e., hydrophilicity or hydrophobicity, ability to bind specific ligands, etc). The advantages of the filtration devices of this type include the uniformity and linearity of the pores and a high ratio between the pores and the matrix.

In accordance into the plastic during manufacture to provide additional tensile strength in dry and wet conditions.

In accordance with yet another technical solution carry out the impregnation of paper and tissues of different nanostructured systems as markers on a fake. In this case, a simple reaction associated with the dye antibodies (e.g., sets, commercially available) can be used to verify the origin of the material. On the other hand, such nanostructured systems can tie dyes or other substances before or after introduction to color paper or fabric, or to modify their appearance or properties of other ways.

Sets

The present invention also provides kits for obtaining nanostructures containing one or more containers chimeras and deletion constructs of the present invention. For example, one such set contains one or more containers cleaned gp35 and purified gp36-34 Chimera. Another such set contains purified gp37-36 Chimera.

The following examples are given to illustrate the present invention and do not limit its scope.

In the examples below, all restrictive enzymes, nucleases, ligase, etc., are commercially available substances from various commercial East is am Bldg. (IUD), Indianapolis, IN.

Example 1

Design, construction and expression of internally deletirovanie P37.

The gene that encodes gp37, contains two sites for restrictase Bgl II, and the first cleavage occurs after nucleotide 293 and after the second nucleotide 1486 (nucleotides are numbered from the initiating methioninamide codon ATG). Thus, cleavage of the DNA fragment that encodes gp37 using Bgl II cut interfering fragment (nucleotides 294-1485) and religioasa 5' and 3' fragments results in the formation of internally deletirovanie gp37, designated as P37, in which arginine-98 is connected to the serine-497.

The reaction mixture for restriction digestion contains:

Gp37 plasmid DNA (1 ág/ál) and 2 ál

NEB buffer # 2 (10X) - 1 ál

H2O - 6 ál

Bgl II (10 units/ál) - 1 ál

Gp37 plasmid is the plasmid pT7-5, in which the gene 37 inserted into the multiple cloning site in a forward direction from the binding site of the ribosome and gene 57 to support the dimerization. The reaction mixture is incubated for 1 hour at 37oC. Then was added 89 μl T4 DNA ligase buffer and 1 µl T4 DNA ligase and the reaction continued under 16oC for 4 hours. Then added tatoonie plasmids, not into fragments using Bgl II in the first stage, reducing their ability to transform about 100 times).

Then the reaction mixture was transformed into E. coli strain BL 21 received from Novagen using standard methods. The transformed mixture was inoculated on nutrient agar containing 100 μg/ml ampicillin, and fees were incubated overnight at 37oC.

Colonies that appeared after incubation over night, sorted and plasmid DNA was extracted and digested with gl II, as described above. Restriction pervari were separated on 1% agarose gels. On successful deletion shows the appearance after gelelectrophoresis new DNA fragment with a size of 4.2 kbp representing nudelatinomen part of the gene 37, which is still attached to the plasmid and which re-forms the site Bgl II by ligating. The DNA fragment size 1.2 kbp associated sites Bgl II in the original gene is no longer present in the plasmid and thus lost from the gel.

Plasmids selected for predicted deletions as described above, transformed into E. coli strain BL21 (DES). Transformants were grown at 30oC as long as the density (A600) key when 30oC for 2 hours, after which the culture was frozen on ice. Then 20 µl of the culture was collected and added to 20 μl of twofold concentrated "craterous (cracking) buffer containing 1% sodium dodecyl sulfate, glycerin and marking dye. 15 μl of this solution was loaded on a 10% polyacrylamide gel; the second aliquot 15 ál of first incubated in a bath of boiling water for 3 minutes and then was placed on the same gel. After electrophoresis the gel was fixed and stained. About expression deletirovanie gp37 be judged by the appearance of species of proteins migrating at values of apparent molecular weight of 65-70,000 daltons in the boiled sample. The degree of dimerization was confirmed by the intensity of the fragments with high molecular weight in recipechannel sample and/or disappearance of the band corresponding to a protein with a mass of 65-70,000 daltons.

The ability deletirovanie polypeptide to the dimerization directly evaluated by testing its ability to recognize anti-P37 by anticorodal, which reacts only with Mature P37-dimers using the standard method of protein Western blot turns.

An alternative analysis on the functional dimerization deletirovanie P37 the ha by the initial induction of the expression of P37, then infection T4 mutant and detection of phage-descendant.

P37 prepared as described above, and found that he is able to complementaly T4 37-phage in vivo.

Example 2

Design, construction and expression of gp37-36 chimeras.

Source plasmid for this design is a system in which the gene that encodes gp37, clone immediately in the opposite direction (i.e. 5') of the gene encoding gp36. This plasmid was digested Hae III, which deleteroom full 3' region gp37 DNA in the forward direction from the nucleotide 724 to the 3'-end, and also removes the 5'-end of the gp36 DNA from the 5'-end to the nucleotide 349. The reaction mixture was identical to the mixture described in example 1, except that used a different plasmid DNA, and the enzyme was a Hae III. Ligation using T4 DNA ligase, bacterial transformation and restriction analysis was carried out as described in Example 1. In this case, cutting the Central part of the gene 37-36 insert and religioasa detects a new plug from 346 vnutriramochnym codons that cut only once under the action of Hae III (after nucleotide 725). Then the resulting construct expressed in E. coli BL21 (DE3) as described in the Example is. This protein will contain the first 242 N-terminal amino acids gp37, merged with the end 104 C-terminal amino acids gp36 (numbered as 118-221). The applicability of such chimeras depends on its ability to timeresults and attached by type end to end. In other words, the carboxy - end of a specified polypeptide has the ability to interact with the amino end of P37 protein dimer of bacteriophage T4 and education attached dimer, and the amino-end dimer specified polypeptide will possess the ability to interact with other specified chimeric polypeptides. This property can be tested by testing for initiating the dimerization and polymerization resulting from the introduction of P37. On the other hand, polyclonal antibodies specific to P36 the dimer can be used for detection P36 after initiation of dimerization using P37.

Chimera gp37-36 was prepared similarly to the methods described above, except that the restriction enzyme TagI used instead of HaeIII. Briefly, 5'-fragment resulting from digestion under the action of TagI gene 37, ligated with the 3'fragment obtained by TagI digestion of the gene 36. Thus, the received design which was residuali E. coli BL21 (DE3) and the Chimera were detected in protein mass CD. It was found that such gp37-36 Chimera inhibits the growth of T4 wild-type in the case when the expression of gp37-36 chimeras to induce infection (in accordance with the analysis on the inhibition of phage in vitro.

Example 3.

Mutation GP37-36 chimeras with obtaining complementary suppressor.

The purpose of this design consists in producing two options dimerizes P37-36 chimeras: one in which the mutated N-terminal polypeptide (A, denoted as*P37-36) and the variant, in which the mutated C-end of the polypeptide (B, denoted as P37-36*). The requirement is that the mutated*P37 N-end is not able to establish a connection with the C-end P36 wild type, but only the N-end of mutated*P36. The main reason for this requirement is that each of A and B is not capable of independent polymerization (which is possible for related P37-36 protein), but can only consistently be associated with each other (i.e., P37-36*+*P37-36 ---> P37-36*--*P37-36).

The second design, *P37-P36*and is generated by recombination*P37-36 P37-36*in vitro. In the case where the monomers*n; similarly*P37 only capable of catalyzing the polymerization of*gp37-36*in (*P37-36*)n. In this case, two such chimeras can be different sizes and different primary sequences with different potential interactions between the side groups and are able to initiate attachment to different surfaces depending on the locking specificity P37.

The original bacterial strain is a Su0the strain of E. coli /who loses the ability to suppression of amber mutations/. In the case when such strain infect mutant T4 bacteriophage containing amber-mutations in genes 35, 36 and 37, replication of the phage is incomplete, because it cannot be synthesized proteins of filamentous outgrowth. If the first strain transformed with a plasmid that directs the expression of genes gp35, gp36 and gp37 wild-type and induce with IPTG and further infect mutant phage, the formed particles of infectious phage, as judged by the appearance of "bitten" colonies. Bitten colony not look round with smooth edges, on the contrary, are systems with missing sectors. This is caused by the impact on microcolony EU create this design 3'-terminal region of the gene g36 (corresponding to C-terminal region gp36) is subjected to mutagenesis with randomly obtained with oligonucleotides. Accidentally received oligonucleotides prepared during chemical synthesis of oligonucleotides by adding trace amounts to several percent/ of the other three nucleotides at a given position so that the resulting oligonucleotide mixture contains a small percentage of incorrect nucleotides at this position. The inclusion of such oligonucleotides in plasmid results in random mutations /with Hutchison al., Methods. Enzymol., 202:356, 1991/.

Mutagenic population of plasmids /containing, however, unmodified genes 36 and 37/ then transformed into su0bacteria and subsequent infection of mutant T4 phage, as described above. In this case, the emergence of non-"bitten" colonies indicates that the C-end-mutated gp36 no longer interacts with P37 wild type with the formation of functional yarns processes. Estimated gp36*the phenotypes found in such non-bitten colonies were checked for the absence of dimeric N-ends on the corresponding immunospecificity, as described above, and positive colonies were used as the source of the plasmids in the next stage.

Several of these mutant plasmids were extracted and subjected to utoro idalou region gp37, prisutstvuushih on the same plasmid. Again /now twice/ methanediamine plasmids transformed into su strain of E. coli and transformants were infected mutant T4 phage. At this stage bacterial tablets were subjected to screening for the repeated appearance of "bitten" colonies. The appearance of such colonies at this stage indicates that the phage subjected to replication by suppressing the non-functional gp36*mutations at the expense*P37 mutations. In other words, these colonies should contain new*P37 polypeptides, which has now acquired the ability to interact with P36*proteins encoded on the same plasmid.

*P37-36 P37-36*paired suppressor Chimera /A and B as defined above is/ are constructed in the same manner as described in example 2. However, in this case, the *P37 used instead P37 wild-type and P36*use instead P36 wild type. Now*P37-36*Chimera can be obtained by restriction *P37-36 P37-36*and religionaries in rekomendowane order. *P37-36*can be mixed with P37-36 Chimera, and polymerization of each of them can be carried out independently in the presence of another system. This can be is.

Design, construction and expression of chimeras gp36-34

Source plasmid for this design is a system in which a vector containing a gene 57 and the gene encoding gp36, cloned directly in the opposite direction (i.e. 5') of the gene encoding gp34. The plasmid was digested with NdeI, which performs the incision after base pair (bp) 219 gene 36 and after a couple reasons 2594 gene 34, thereby carrying out a deletion end 148 C-terminal codons of the gp36 fragment and the first 865 N-terminal codons of the fragment gp34. The reaction mixture identical to that described in example 1, except that used a different plasmid DNA, and the enzyme used was a NdeI /NEB/. Ligation using T4 DNA ligase, bacterial transformation and restriction analysis was also carried out as described in Example 1. The result has been a new hybrid gene that encodes a protein of 497 amino acids (73 N-terminal amino acids gp36 and 424 C-terminal amino acids gp34, numbered as 866-1289).

Alternatively, the original plasmid was cut with SphI on a pair of bases 648 in the gene 34 and deletion set Exo-Size /NEB/ used to implement the described you and with methods described in Example 1. Successful expression of gp36-34 Chimera to be judged by the appearance of the protein product of approximately 55.000 daltons. Preferably, the amino ends of the polypeptide of glycosilated had the ability to interact with protein gp35, and then carboxy-ends will have the ability to interact with other attached gp35 molecules. The successful formation of the dimer can be detected by the reaction with anti-P36 antibody, or accession, gp35, or analysis on the inhibition of phage in vitro as described in Example 2.

Example 5.

The allocation of thermolabile proteins for self-Assembly.

Thermolabile patterns can be applied in nanostructures for:

a) initiating polymerization of the Chimera /for example, gp37-36/ at low temperature and inactivation and separation from the initiator at high temperature; (b) iniciirovanie education angle between P36 and gp35 /for example, options gp35 with thermolabile sites of attachment for P36 N-ends or P34 C, option P36, forming a temperature-sensitive accession to gp35 and P34 with thermolabile C-terminal site of joining/. Thermolability may be reversible, allowing re-attach relevant about the options gp37, providing thermoinsulating separation P36-P37 connection, the 5'-end gp37 DNA is subjected randomizearray mutagenesis with the use of oligonucleotides as described above. Then the fragment mutagenicities DNA recombinable in T4 phage by infection of the cell containing mutagenically DNA, T4 phage, containing two amber mutations flanking region of mutagenesis. After disconneting infection alberny phage were subjected to selection at low temperature on E. coli at 30o. The offspring of such plaques resuspendable in the buffer and subjected to stimulation by heating at 60oC. At this temperature the thread ridge of the wild type remain intact and functional, while thermolabile version allocate terminal P37 cells and thereby make such a non-infectious phage.

At this stage, the wild-type phage are removed: 1) adsorption of phage wild-type sensitive bacteria and deposition (or filtering) bacteria adsorbed phage wild type; or (2) reaction of the lysate with anti-P37 antibody and then with immobilized protein a and removing the adsorbed phage wild type. As a result of implementation of any way particles needley. Non-infectious phage with the lack of terminal P37 fragments /and probably also the rest of the processes/ next treated with 6M urea and mixed with bacterial spheroplasts to ensure infection low multiplicity, with the result that they are replicated at a low temperature, and produce offspring. On the other hand, infectious phages reconstructed by in vitro incubation of mutant phage in the presence P37 wild-type at 30oC; then carry the infection of intact bacterial cells using standard procedures. As a result of implementation of the last of the above methods of infection carry out specific selection of mutant phage in which thermolability connection P36-P37 is reversible.

In either method, the phage population is subjected to repeated selection as described above, after which individual phage particles emit trombotsitnoy clearance under 30oC. Finally, putative mutants individually evaluated according to the following characteristics: 1) loss of infection after incubation at high temperatures /40-60oC/, which is measured by the decrease in titer; 2) loss P37 after mi phage; and 3) morphological changes in the threads processes after incubation at high temperature, which was evaluated by electron microscopy.

After isolation of mutants and their phenotypes P37 gene were subjected to sequencing. If mutations are localized in specific regions or residues, such sequences are targeted for siteprovides mutagenesis to optimize the desired characteristics.

Finally, the mutant gene 37 clone into expression plasmids and individually Express in E. coli as described in Example 1. Then mutant P37 dimers purified from bacterial extracts and used in reactions Assembly in vitro.

Similarly can be selected such mutant gp35 polypeptides that exhibit temperature-sensitive interaction with the N-end P36 or C-end P34. For thermolabile interactions with P34 phage incubated at high temperature, resulting in loss of the entire distal half of the thread ridge /ie gp35-P36-P37/. In this case, the difference in the experimental procedure consists in the following: 1) randomized mutagenesis performed on the full gp35 gene; 2) the wild-type phage (and distal polonici Ter the e (but still containing attached near polonici process) by deposition as the distal polonica, and of phage particles containing intact filamentous appendages, with any of the antibodies antidotale half the threads of the process, followed by pellets staphylococcal A protein; 3) the mutant phage remaining in the supernatant, reactivit by incubation at low temperature in the presence of bacterial extracts containing the intact distal polonici wild type; and 4) a series of temperature-sensitive mutants of the gene 35, grown at 30oC can be tested for reversible thermolability by inactivation at 60oC and reincubate at 30oC. the Inactivation is carried out on concentrated suspensions of phage, and reincubate at 30oC is carried out before or after dilution. If the phage successfully reactivated before, but not after dilution, it indicates that it gp35 is reversible heat-sensitive.

To obtain mutations of the gene 36 with thermolabile communication gp35-P36 C-end of the gene 36 described above is subjected to mutagenesis and mutant subjected to selection on reversibility. The alternative is that this procedure mutagenesis gp35 to obtain mutant gene 35, in which the relationship gp35-P36 dissociates at 60oC. In this case, incubation in the presence of anti-gp35 antibodies can be used to precipitate the phage is ECU variant gp35 remains attached to P34.

Example 6.

Assembly of one-dimensional rods.

A. Simple Assembly: P37-36 dimer described in Example 2, capable of self-Assembly, but requires the presence P37 initiator to associate the first unit of the rod. Therefore, P37 or P37 dimer or attached to a solid matrix, or they are in free form in solution to perform the functions of the initiator. If you use the initiator attached to a solid matrix, it is preferable to use thermolabile P37 dimer. Add extract containing gp37-36, or purified gp37-36 chimeras resulted in the Assembly line multimers of increasing length. In the case of bonding with the matrix, end the rods are released by brief incubation at high temperature /40-60oC/ depending on the characteristics of the specific thermolabile P37 variants/.

The ratio between the initiator and gp37-36 may change the distribution of rods by size measure any of the following ways:

1/ using gel chromatography;

2/ measurement increasing the viscosity of the solution; and

3/ direct measurement by electron microscopy.

B. Stage Assembly: P37-36*P37-36 P37-36, described in Primay according to the following procedure:

1. The initiator P37 /preferably thermolabile/ attach to the matrix.

2. Add an excess of*p37-36 for connection and oligomerization in the form P37-36 komolgorov to the N-end P37.

3. Wash off unreacted P37-36*and the system is poured excessive amounts of*p37-36.

5. Repeat stages 2-4 n-1 times.

6. Release the Assembly from the matrix by short incubation at high temperature, as described above.

The linear sizes of protein bars in the party will depend on the length of a single heterodimer and number of cycles / n / attachment. The advantage of this method is ensuring absolute reproducibility of the rod length and homogeneous, monodisperse distribution by size when switching from one drug to another.

Example 7.

Stage Assembly of polygons.

The following strategies assemblies use gp35 as gussets, providing education polygons. For the purposes of this example it is assumed that the angle formed by the gp35, is 137o. Rod cell contains P36-34 Chimera described in Example 4, which is not capable of semipolitical. P36-34 homodimerization R36-34 P36-34.

1. Initiator: Incomplete distal polunicheva P36-37 was attached to the solid matrix by using P37 C-Terminus. Then added thermolabile gp35 as described in Example 5 with the formation of the intact initiator.

2. Excessive P36-34 Chimera was added to attach the unit P36-34. After bonding with the matrix via gp35 unrelated Chimera laundered.

3. Then, excess) was added gp35 wild-type /ie determinability/. After incubation, unbound material was washed.

4. Stage 2 and 3 were repeated 7-8 times.

5. The Assembly is freed from the matrix by a short incubation at high temperature.

Released polymer rod length 8 units will form a regular 8-sided polygon, the sides of which contain P36-34 dimer and its compounds containing monomer gp35 wild type. However, among these 8 units, connected in the form of spirals, there are several multimeric. If the unit is not closed, but instead attaches the other end, it is no longer able to withdraw and the spiral can be created in any direction. The direction of the first overlapping determines the chiral dosimetry spiral. The rods out of ten /or so/or 128.6o/. The probability of closure of the regular polygon depends not only on the mean angle gp35, but also from its flexibility, which can optionally be controlled through genetic modification or the modification of the environment.

The type of polygon that is formed by use of such procedures depends on the length of the rod cells and the angle formed by the gusset. For example, alternating core units of various sizes can be used in stage 2. In addition, can be used variant gp35 polypeptides that form angles other than the natural angle 137othat allows you to get various regular /correct/ polygons. In addition, for a given polygon with an even number of sides and equal angles, the parties to any half can be of any size, provided the symmetry of the two halves.

1. The selected polypeptide to obtain nanostructures, representing a variant of gp36 protein of bacteriophage T4 (SEQ ID NO:5), do not possess the ability to interact with aminocom.com P37 protein oligomer of the bacteriophage T4 due to mutations in the gene region 36 corresponding To the end region gp36.

3. The selected protein to obtain nanostructures, including the first part of the gp36 protein of T-even-like bacteriophage consisting of the first 10 - 60 N-terminal amino acids gp36 protein of SEQ NO:5, merged with the second part of protein gp34 T-even-like bacteriophage, which consists of 10 - 60 C-terminal amino acid protein gp34 SEQ ID NO:2.

4. Isolated protein to obtain a nanostructure comprising at least 20 contiguous amino acids gp37 protein of T-even-like bacteriophage having the amino acid sequence of SEQ ID NO:6, and which lacks at least 5 amino acids of the amino - and carboxy end of the protein.

5. Isolated protein to obtain a nanostructure comprising at least 20 contiguous amino acids gp36 protein of T-even-like bacteriophage having the amino acid sequence of SEQ ID NO:5, and in which there are no, at least 5 amino acids of the amino - and carboxy end of the protein.

6. Isolated protein to obtain nanostructures, including, at the very the awn SEQ ID NO:2, and which lacks at least 5 amino acids of the amino - and carboxy end of the protein.

 

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