Genetic engineering of proteins, hybrid proteins, viral vectors. Reporter proteins in fusion proteins

After considering how to generate site-specific mutations, it takes only one step to find yourself face to face with the rapidly developing field of molecular genetics called protein engineering. Indeed, the development of methods of targeted mutagenesis has made it possible not only to modify individual proteins with high precision and study their structural-functional relationships, but also to construct new proteins that did not exist in nature. Impressive results of this approach are hybrid proteins obtained by combining fragments and functional domains of different polypeptide chains using genetic engineering methods.

Another promising area of protein engineering is the design of biologically active peptides with pharmacological activity.

Peptide and Epitope Libraries

In a living organism, most biological processes are controlled through specific protein-protein or protein-nucleic acid interactions. Such processes include, for example, the regulation of gene transcription under the influence of various protein factors, the interaction of protein ligands with receptors on the surface of cells, as well as the specific binding of antigens by corresponding antibodies. Understanding the molecular mechanisms of interaction of protein ligands with receptors is of great fundamental and applied importance. In particular, the development of new protein drugs usually begins with the identification of the initial amino acid sequence that has the required biological activity (the so-called “lead” sequence). However, peptides with a basic amino acid sequence may also have undesirable biological properties: low activity, toxicity, low stability in the body, etc.

Before the advent of peptide libraries, improvement of their biological properties was carried out by sequential synthesis of a large number of analogs and testing of their biological activity, which required a lot of time and money. In recent years, it has become possible to create thousands of different peptides in a short time using automatic synthesizers. The developed methods of targeted mutagenesis have also made it possible to dramatically expand the number of proteins obtained simultaneously and sequentially tested for biological activity. However, only recently developed approaches to creating peptide libraries have produced the millions of amino acid sequences required to effectively screen for peptides that best meet the criteria. Such libraries are used to study the interaction of antibodies with antigens, obtain new enzyme inhibitors and antimicrobial agents, design molecules with the desired biological activity, or impart new properties to proteins, such as antibodies.

Based on the methods of preparation, peptide libraries are divided into three groups. The first group includes libraries obtained using chemical synthesis of peptides, in which individual peptides are immobilized on microcarriers. With this approach, after the addition of successive amino acids in individual reaction mixtures to peptides immobilized on microcarriers, the contents of all reaction mixtures are combined and divided into new portions, which are used at the next stage of addition of new amino acid residues. After a series of such steps, peptides are synthesized containing the sequences of amino acids used in the synthesis in all sorts of random combinations.

Libraries of peptides immobilized on microcarriers have a significant drawback: they require the use of purified receptors in soluble form during screening. At the same time, in most cases, membrane-associated receptors are most often used in biological tests carried out for basic and pharmacological research. According to the second method, peptide libraries are obtained using solid-phase peptide synthesis, in which at each stage of the chemical addition of the next amino acid to the growing peptide chains, equimolar mixtures of all or some precursor amino acids are used. At the final stage of synthesis, the peptides are separated from the carrier, i.e. converting them into soluble form. The third approach to constructing peptide libraries, which we are now describing, became feasible precisely thanks to the development of genetic engineering methods. It perfectly illustrates the capabilities of such methods and is undoubtedly a major advance in their application. In this regard, let us consider in more detail the results of using peptide libraries in the study epitopes(antigenic determinants) proteins.

Genetic engineering technology for producing hybrid proteins has made it possible to develop an effective method for producing short peptides for analyzing their biological activity. As in the case of gene libraries, peptide libraries obtained by genetic engineering methods represent a large (often exhaustive) set of short peptides. Two recent observations make it possible to consider a library of peptides simultaneously and as a library of protein epitopes. First, short peptides can include all the essential amino acid residues that play a major role in antibody interaction, and they are able to mimic large antigenic determinants of proteins. Second, in most cases, noncovalent bonds formed between the few most important amino acid residues of protein ligands and their receptors make a major contribution to the total energy of the ligand-receptor interaction. With this in mind, any peptide can be considered a potential ligand, hapten, or part of the antigenic determinant of larger polypeptides, and any peptide library can be considered a library of protein epitopes or potential ligands for the corresponding protein receptors.

Rice. II.19. Scheme of expression of peptide epitopes on the surface of the shell of filamentous coliphages

Peptide epitopes are located in the hybrid polypeptide chains of the minor protein pIII ( A) or the basic protein pVIII of the viral envelope ( b). Arrows indicate the position of oligonucleotide fragments encoding epitopes in the bacteriophage genome, as well as the position of the epitopes themselves. As part of the polypeptide pIII ( A) only one copy of the epitope is shown (in fact, their number reaches 4–5)

The peptide library obtained as a result of the implementation of the third approach, in its modern form, is a set of tens or even hundreds of millions of short different amino acid sequences that are expressed on the surface of bacteriophage virions as part of their own structural proteins. This becomes possible thanks to the introduction of hybrid recombinant genes encoding altered structural proteins of its virions into the genome of bacteriophages using genetic engineering methods. (This method is known as phage display.) As a result of the expression of such genes, hybrid proteins are formed, at the N- or C-termini of which (see below) additional amino acid sequences are present. The most well-developed system for constructing peptide libraries using genetic engineering methods uses the small filamentous coliphage f1 and its two proteins: the major and minor coat proteins pVIII and pIII. In vivo, both proteins are synthesized as polypeptide chains with short N-terminal signal sequences that are cleaved off by signal peptidase during their maturation after transfer to the interior of the bacterial membrane. Mature proteins are integrated into the bacteriophage shell during its assembly. In this case, the pVIII protein forms the main shell of the bacteriophage, while four or five pIII molecules are associated with the terminal part of the virion and ensure the interaction of viral particles with the genital villi of E. coli cells (Fig. II.19). Using genetic engineering methods, peptides are combined with proteins - directly with their N-terminal sequences or at a short distance from them. The terminal sequences of most proteins are more flexible and, as a rule, are exposed on the surface of the globule, which makes it possible to obtain hybrid recombinant proteins without significantly disrupting their basic properties, and also makes the integrated peptides accessible for recognition from the outside. In addition, in this position, the spatial structure of the peptides themselves is less influenced by the carrier protein. During the experiments, it was found that the introduction of foreign peptides into the N-terminal part of the pIII protein does not have a significant effect on the viability and infectivity of phage particles, while the combination of peptides >5 amino acid residues long with the N-terminal part of the pVIII protein disrupts the assembly of virions. The last difficulty can be overcome by delivering wild-type pVIII protein molecules to the site of virion assembly, the synthesis of which is directed by the corresponding gene of the helper virus. In this case, the bacteriophage shell will contain both modified pVIII proteins and wild-type polypeptides from the helper virus.

Rice. II.20. Scheme for constructing a recombinant viral genome containing inserts of degenerate oligonucleotides to obtain a library of epitopes

Double-stranded oligonucleotide ( A), containing degenerate NNK codons and the same restriction sites as part of the linkers, are ligated with the DNA of the Fuse5 vector ( b), digested by restriction enzyme Sfi I, with the formation of a recombinant genome ( V), which directs the synthesis of a hybrid recombinant protein ( G), containing at the N-terminus the indicated amino acid sequence

When constructing a peptide library, first of all, two oligonucleotides complementary to each other are synthesized, which after annealing form a double-stranded molecule, the central part of which encodes the peptides themselves (Fig. II.20, A), and the single-stranded sections protruding at the ends are complementary to the “sticky” ends of the vector, obtained under the action of the corresponding restriction enzyme (see Fig. II.20, b).

To encode amino acids of peptides, degenerate codons of the type NNK or NNS are used, which include all four nucleotides (N) in the first and second positions, G or T (K), and G or C (S) in the third position. With this approach, information about all 20 amino acids and one stop codon is contained in 32 different NNK and NNS codons, rather than 64, as is the case in the natural genetic code.

In the process of synthesizing degenerate oligonucleotides encoding the peptides under study, at each stage individual nucleotides are used for codons of invariant amino acids flanking the variable region of the peptide, as well as equimolar mixtures of nucleotides for regions encoding random sequences. The resulting set of degenerate oligonucleotides is then cloned in the form of single-stranded fragments in the corresponding sites of the bacteriophage coat protein gene as part of the phage vector or phasmid. Alternatively, for such a set of oligonucleotides (chemically or using PCR), complementary strands are synthesized with the inclusion of inosine in the variable regions, since its residues are known to pair with the C and T bases of the template, which facilitates the formation of correct duplexes between the corresponding oligonucleotides. The resulting double-stranded oligonucleotides, if necessary, are treated with appropriate restriction enzymes and cloned in a phage vector. The resulting recombinant molecules (see Fig. II.20, V) DNA is introduced into bacterial cells, obtaining ~10 9 transformants per 1 mg of recombinant DNA, the resulting phage particles are propagated in bacteria and, after purification, are examined for the presence of recombinant peptides (see Fig. II.20, G), capable of interacting with the studied receptors in the proteins of their virions.

The number of individual phage clones in the library is decisive for its use. For example, a library containing all possible hexapeptides should contain 64 million (20 6) different six-membered amino acid sequences encoded by ~1 billion (32 6) different hexacodons (32 is the number of codons with which any of the 20 amino acids can be encoded using the method proposed above, namely using codons NNK or NNS). To solve such a problem, very large libraries must be obtained containing at least 2 10 8 - 3 10 8 individual, independent clones, and the value of 10 9 is currently the upper limit on the number of individual library clones that can still be obtained. practically use.

Based on this, we can conclude that the maximum length of peptides, including all possible combinations of 20 amino acids, which can be worked with using peptide libraries, is 6 amino acid residues. However, it should be kept in mind that a library of 15-member peptides of the same size (2–3 × 10 8 clones) will contain more diverse hexapeptides than the library of 6-member peptides discussed above. In addition, since only a limited number of amino acid residues in a peptide actually determine its biological activity, a library of 15-mer peptides may be more representative than a library of shorter peptides with the same number of clones.

Rice. II.21. Scheme for selecting phage particles possessing the required epitopes

Three recombinant phage particles expressing different epitopes within pIII are shown. Only the epitope of the central phage particle is recognized by the biotinylated antibody molecule immobilized on a Petri dish using streptavidin and used for library screening

In order to isolate peptides with the desired biological activity from a library, various screening methods are used. In particular, to isolate peptides that mimic certain epitopes, biotinylated monoclonal antibodies of appropriate specificity are used, which are immobilized on a solid support using streptavidin (Fig. II.21). Phage particles expressing the corresponding epitopes on their surface interact with antibodies and are retained by the substrate, while other recombinant phage particles are removed during the washing process. The phage particles retained on the substrate are then eluted with acid, individual clones are further propagated in bacterial cells, and the epitopes expressed on them are examined according to various criteria. The presence of identical or similar nucleotide sequences among the cloned sequences indicates the specificity of the purification process. Individual clones are then characterized by other methods, in particular enzyme immunoassays. At the final stage of the study, the isolated peptides are synthesized and comprehensively studied in a purified state.

Currently, there is evidence of some work carried out using peptide libraries. In one of these studies, peptides were isolated from a library whose amino acid sequence differed sharply from the amino acid sequence of the true epitope of the antigen under study. However, such a peptide bound strongly to specific antibodies and competed for binding to the natural antigen. This allowed us to conclude that there is a possibility of existence mimotopes– short peptides that imitate natural epitopes, the amino acid sequences of which differ significantly from each other. It was possible to establish the canonical amino acid sequences of peptides that imitate epitopes of natural proteins, and among them to identify amino acid residues that play a key role in the antigen-antibody interaction.

One of the promising applications of peptide libraries is the identification of peptide ligands that mimic “structural” epitopes formed on the surface of protein globules as a result of folding of their polypeptide chains, which is accompanied by spatial proximity of amino acid residues located in the polypeptide chain at a considerable distance from each other. Using peptide libraries, it is possible to identify peptide analogues of various non-protein epitopes. Apparently, in the near future it will be possible to use peptide libraries to obtain new drugs, create diagnostic tools, and produce effective vaccines. In the field of designing new drugs, research efforts could be aimed at creating peptide ligands that specifically interact with receptors of biomedical interest. Knowledge of the structure of such ligands would simplify the preparation of non-protein drugs on this basis.

Libraries of peptides and epitopes will also find their use in studies of the mechanisms of the humoral immune response, as well as diseases of the immune system. In particular, most autoimmune diseases are accompanied by the formation of autoantibodies against antigens of the body's own. These antibodies in many cases serve as specific markers of a particular autoimmune disease. Using a library of epitopes, in principle, it is possible to obtain peptide markers, with the help of which it would be possible to monitor the specificity of autoantibodies during the development of a pathological process both in an individual organism and in a group of patients and, in addition, to determine the specificity of autoantibodies in diseases of unknown etiology .

Libraries of peptides and epitopes can also potentially be used for screening immune sera to identify peptides that specifically interact with protective antibodies. Such peptides will mimic the antigenic determinants of pathogenic organisms and serve as targets for the body's protective antibodies. This will allow the use of such peptides for vaccination of patients who lack antibodies against the corresponding pathogens. The study of epitopes using peptide libraries is a special case of one of the many areas of their use in applied and fundamental studies of the interaction of ligands and receptors. Further improvement of this approach should facilitate the creation of new drugs based on short peptides and be useful in fundamental studies of the mechanisms of protein-protein interactions.

Course work

discipline: Agricultural biotechnology

on the topic: “Protein engineering”

Introduction. Protein engineering

1 The concept of protein engineering. History of development

2 Protein engineering strategies. Examples of engineered proteins. Applications of Protein Engineering

1 Peptide and epitope libraries

2 Reporter proteins in fusion proteins

3 Some achievements of protein engineering.

Conclusion

Bibliography

Essay

Topic: Protein engineering.

Key words: biotechnology, genetic engineering, protein, genetic code, gene, DNA, RNA, ATP, peptides, epitope.

The purpose of the course work: to study the concept of “protein engineering” and the potential possibilities of its use.

Potential opportunities of protein engineering:

By changing the strength of binding of the substance being converted - the substrate - to the enzyme, it is possible to increase the overall catalytic efficiency of the enzymatic reaction.

By increasing the stability of the protein over a wide range of temperatures and acidity, it can be used under conditions under which the original protein denatures and loses its activity.

By creating proteins that can function in anhydrous solvents, it is possible to carry out catalytic reactions under non-physiological conditions.

By changing the catalytic center of an enzyme, it is possible to increase its specificity and reduce the number of unwanted side reactions

By increasing the protein's resistance to enzymes that break it down, the purification procedure can be simplified.

By modifying a protein so that it can function without its usual non-amino acid component (vitamin, metal atom, etc.), it can be used in some continuous technological processes.

By changing the structure of the regulatory regions of the enzyme, it is possible to reduce the degree of its inhibition by the product of the enzymatic reaction according to the type of negative feedback and thereby increase the yield of the product.

It is possible to create a hybrid protein that has the functions of two or more proteins.

It is possible to create a hybrid protein, one of the sections of which facilitates the release of the hybrid protein from the cultured cell or its extraction from the mixture.

Introduction

Since time immemorial, biotechnology has been used mainly in the food and light industries: in winemaking, bakery, fermentation of dairy products, in the processing of flax and leather, based on the use of microorganisms. In recent decades, the possibilities of biotechnology have expanded enormously. This is due to the fact that its methods are more profitable than conventional ones for the simple reason that in living organisms, biochemical reactions catalyzed by enzymes occur under optimal conditions (temperature and pressure), are more productive, environmentally friendly and do not require chemical reagents that poison the environment.

The objects of biotechnology are numerous representatives of groups of living organisms - microorganisms (viruses, bacteria, protozoa, yeasts), plants, animals, as well as cells isolated from them and subcellular components (organelles) and even enzymes. Biotechnology is based on physiological and biochemical processes occurring in living systems, which result in the release of energy, synthesis and breakdown of metabolic products, and the formation of chemical and structural components of the cell.

The main direction of biotechnology is the production, using microorganisms and cultured eukaryotic cells, of biologically active compounds (enzymes, vitamins, hormones), medications (antibiotics, vaccines, serums, highly specific antibodies, etc.), as well as valuable compounds (feed additives, for example, essential amino acids, feed proteins, etc.).

Genetic engineering methods have made it possible to synthesize in industrial quantities hormones such as insulin and somatotropin (growth hormone), which are necessary for the treatment of human genetic diseases.

Biotechnology solves not only specific problems of science and production. It has a more global methodological task - it expands and accelerates the scale of human impact on living nature and promotes the adaptation of living systems to the conditions of human existence, i.e. to the noosphere. Biotechnology, thus, acts as a powerful factor in anthropogenic adaptive evolution.

Biotechnology, genetic and cell engineering have promising prospects. As more and more new vectors appear, people will use them to introduce the necessary genes into the cells of plants, animals and humans. This will make it possible to gradually get rid of many hereditary human diseases, force cells to synthesize the necessary drugs and biologically active compounds, and then directly proteins and essential amino acids used in food. Using methods already mastered by nature, biotechnologists hope to obtain hydrogen through photosynthesis - the most environmentally friendly fuel of the future, electricity, and convert atmospheric nitrogen into ammonia under normal conditions.

The physical and chemical properties of natural proteins often do not satisfy the conditions under which these proteins will be used by humans. A change in its primary structure is required, which will ensure the formation of a protein with a different spatial structure than before and new physicochemical properties, allowing it to perform the functions inherent in natural protein under other conditions. Protein engineering deals with the construction of proteins.

Another area of application of protein engineering is the creation of proteins that can neutralize substances and microorganisms that can be used for chemical and biological attacks. For example, hydrolase enzymes are capable of neutralizing both nerve gases and pesticides used in agriculture. Moreover, the production, storage and use of enzymes is not dangerous to the environment and human health.

To obtain an altered protein, combinatorial chemistry methods are used and directed mutagenesis is carried out - introducing specific changes to the coding sequences of DNA, leading to certain changes in amino acid sequences. To effectively design a protein with desired properties, it is necessary to know the patterns of formation of the spatial structure of the protein, on which its physicochemical properties and functions depend, that is, it is necessary to know how the primary structure of the protein, each of its amino acid residues affects the properties and functions of the protein. Unfortunately, for most proteins the tertiary structure is unknown; it is not always known which amino acid or sequence of amino acids needs to be changed in order to obtain a protein with the desired properties. Already now, scientists using computer analysis can predict the properties of many proteins based on the sequence of their amino acid residues. Such an analysis will greatly simplify the procedure for creating the desired proteins. In the meantime, in order to obtain a modified protein with the desired properties, they mainly go in a different way: they obtain several mutant genes and find the protein product of one of them that has the desired properties.

Various experimental approaches are used for site-directed mutagenesis. Having received the modified gene, it is integrated into a genetic construct and introduced into prokaryotic or eukaryotic cells that synthesize the protein encoded by this genetic construct.

I. Protein engineering

1 The concept of protein engineering. History of development

Protein engineering is a branch of biotechnology that deals with the development of useful or valuable proteins. This is a relatively new discipline that focuses on the study of protein folding and the principles of protein modification and creation.

There are two main strategies for protein engineering: directed protein modification and directed evolution. These methods are not mutually exclusive; researchers often use both. In the future, more detailed knowledge of protein structure and function, as well as advances in high technology, may significantly expand the possibilities of protein engineering. As a result, even unnatural amino acids can be incorporated thanks to a new method that allows new amino acids to be incorporated into the genetic code.

Protein engineering originated at the intersection of protein physics and chemistry and genetic engineering. It solves the problem of creating modified or hybrid protein molecules with specified characteristics. A natural way to implement such a task is to predict the structure of the gene encoding the altered protein, carry out its synthesis, cloning and expression in recipient cells.

The first controlled protein modification was carried out in the mid-60s by Koshland and Bender. To replace the hydroxyl group with a sulfhydryl group in the active site of the protease, subtilisin, they used a chemical modification method. However, as it turned out, such thiolsubtilisin does not retain protease activity.

Chemically, a protein is a single type of molecule, which is a polyamino acid chain or polymer. It is composed of amino acid sequences of 20 types. Having learned the structure of proteins, people asked the question: is it possible to design completely new amino acid sequences so that they perform the functions humans need much better than ordinary proteins? The name Protein Engineering was appropriate for this idea.

People began to think about such engineering back in the 50s of the 20th century. This happened immediately after deciphering the first protein amino acid sequences. In many laboratories around the world, attempts have been made to duplicate nature and chemically synthesize given absolutely arbitrary polyamino acid sequences.

The chemist B. Merrifield succeeded most in this. This American managed to develop an extremely effective method for the synthesis of polyamino acid chains. For this, Merrifield was awarded the Nobel Prize in Chemistry in 1984.

Figure 1. Scheme of how protein engineering works.

The American began to synthesize short peptides, including hormones. At the same time, he built an automaton - a “chemical robot” - whose task was to produce artificial proteins. The robot caused a sensation in scientific circles. However, it soon became clear that his products could not compete with what nature produces.

The robot could not accurately reproduce the amino acid sequences, that is, it made mistakes. He synthesized one chain with one sequence, and the other with a slightly modified one. In a cell, all molecules of one protein are ideally similar to each other, that is, their sequences are absolutely identical.

There was another problem. Even those molecules that the robot synthesized correctly did not take on the spatial form necessary for the enzyme to function. Thus, the attempt to replace nature with the usual methods of organic chemistry led to very modest success.

Scientists could only learn from nature, looking for the necessary modifications of proteins. The point here is that in nature there are constantly mutations leading to changes in the amino acid sequences of proteins. If you select mutants with the necessary properties that process a particular substrate more efficiently, then you can isolate from such a mutant an altered enzyme, thanks to which the cell acquires new properties. But this process takes a very long period of time.

Everything changed when genetic engineering appeared. Thanks to her, they began to create artificial genes with any nucleotide sequence. These genes were inserted into prepared vector molecules and the DNA was introduced into bacteria or yeast. There, a copy of RNA was taken from the artificial gene. As a result, the required protein was produced. Errors in its synthesis were excluded. The main thing was to select the right DNA sequence, and then the cell’s enzyme system itself did its job flawlessly. Thus, we can conclude that genetic engineering has opened the way to protein engineering in its most radical form.

1.2 Protein engineering strategies

Targeted protein modification. In targeted protein modification, the scientist uses detailed knowledge of the protein's structure and function to make the desired changes. In general, this method has the advantage of being inexpensive and technically uncomplicated, since the technique of site-directed mutagenesis is well developed. However, its main disadvantage is that information about the detailed structure of a protein is often lacking, and even when the structure is known, it can be very difficult to predict the effect of various mutations.

Protein modification software algorithms strive to identify new amino acid sequences that require little energy to form a predefined target structure. While the sequence that must be found is large, the most difficult requirement for protein modification is a fast, yet precise, way to identify and define the optimal sequence, as opposed to similar suboptimal sequences.

Directed evolution. In directed evolution, random mutagenesis is applied to a protein and selection is made to select variants that have certain qualities. Next, more rounds of mutation and selection are applied. This method mimics natural evolution and generally produces superior results for directed modification.

An additional technique known as DNA shuffling mixes and identifies parts of successful variants to produce better results. This process mimics the recombinations that occur naturally during sexual reproduction. The advantage of directed evolution is that it does not require prior knowledge of protein structure, nor is it necessary to be able to predict what effect a given mutation will have. Indeed, the results of directed evolution experiments are surprising because the desired changes are often caused by mutations that should not have such an effect. The disadvantage is that this method requires high throughput, which is not possible for all proteins. Large quantities of recombinant DNA must be mutated and the products must be screened for the desired quality. The sheer number of options often requires the purchase of robotics to automate the process. In addition, it is not always easy to screen for all qualities of interest.

II. Examples of engineered proteins

Protein engineering can be based on chemical modification of a finished protein or on genetic engineering methods that make it possible to obtain modified versions of natural proteins.

The design of a specific biological catalyst is carried out taking into account both the specificity of the protein and the catalytic activity of the organometallic complex. Here are examples of such modification carried out to obtain “semi-synthetic bioorganic complexes”. Sperm whale myoglobin is capable of binding oxygen, but does not have biocatalytic activity. As a result of the combination of this biomolecule with three electron-transfer complexes containing ruthenium, which bind to histidine residues on the surface of protein molecules, a complex is formed that is capable of reducing oxygen while simultaneously oxidizing a number of organic substrates, such as ascorbate, at a rate almost the same as for natural ascorbate oxidase. In principle, proteins can be modified in other ways. Consider papain, for example. It is one of the well-studied proteolytic enzymes for which a three-dimensional structure has been determined. Near the cysteine-25 residue on the surface of the protein molecule there is an extended groove in which the proteolysis reaction occurs. This site can be alkylated by a flavin derivative without changing the accessibility of the potential substrate binding site. Such modified flavopapains were used for the oxidation of M-alkyl-1,4-dihydronicotinamides, and the catalytic activity of some of these modified proteins was significantly higher than that of natural flavoprotein-NADH dehydrogenases. Thus, it was possible to create a very effective semi-synthetic enzyme. The use of flavins with highly active, positioned electron-withdrawing substituents may make it possible to develop effective catalysts for the reduction of nicotine amide.

Major advances achieved recently in the chemical synthesis of DNA have opened up fundamentally new opportunities for protein engineering: the design of unique proteins that do not occur in nature. This requires further development of technology, so that changing genes using genetic engineering methods leads to predictable changes in proteins, to an improvement in their well-defined functional characteristics: turnover number, Km for a specific substrate, thermal stability, temperature optimum, stability and activity in non-aqueous solvents, substrate and reaction specificity, requirement for cofactors, pH optimum, protease resistance, allosteric regulation, molecular weight and subunit structure. Typically, such improvement has been achieved through mutagenesis and selection, and more recently through chemical modification and immobilization. To successfully design a specific type of protein molecule, it is necessary to identify a number of fundamental patterns connecting the structural features of proteins and their desired properties. Thus, knowing the exact crystal structure of the protein molecule under study, it is possible to identify those parts of it that should be specifically modified to increase its catalytic activity. Such a modification may consist of changing the amino acid sequence of the protein.

Another example is the implementation of site-specific mutagenesis. It happens as follows. The gene for the protein that interests the researcher is cloned and inserted into a suitable genetic carrier. Then an oligonucleotide primer with the desired mutation is synthesized, the sequence of which, consisting of ten to fifteen nucleotides, is sufficiently homologous to a certain region of the natural gene and is therefore capable of forming a hybrid structure with it. This synthetic primer is used by polymerases to initiate the synthesis of a complementary copy of the vector, which is then separated from the original and used for controlled synthesis of the mutant protein. An alternative approach is based on cleavage of the chain, removal of the site to be changed and its replacement with a synthetic analogue with the desired nucleotide sequence.

Tyrosyl-tRNA synthetase catalyzes the aminoacylation reaction of tyrosine tRNA, which involves activation of tyrosine by ATP to form tyrosyl adenylate. The gene for this enzyme, isolated from Bacillus stearothermophilus, was inserted into bacteriophage M13. The catalytic properties of the enzyme, especially its ability to bind substrate, were then altered by site-specific modification. Thus, threonine-51 was replaced by alanine. This resulted in a twofold increase in substrate binding, apparently due to the inability to form a hydrogen bond between this residue and the tyrosyl adenylate. When replacing alanine with proline, the configuration of the enzyme molecule is disrupted, but the ability to bind the substrate increases a hundredfold, since its interaction with histidine-48 is facilitated. Similar site-specific changes have been obtained in β-lactamase, and they are usually accompanied by inactivation of the enzyme. Replacing serine-70 with cysteine leads to the formation of p-thiol lactamase, the binding constant of which does not differ from that of the natural enzyme, but the activity towards penicillin is only 1-2%. Nevertheless, the activity of this mutant enzyme against some activated cephalosporins is no less than the original activity or even exceeds it; these proteins are also more resistant to proteases.

Site-specific mutations are now used to test the adequacy of structural studies. In some cases, they were able to show that the structural stability of a protein and its catalytic activity can be decoupled. A sufficient amount of information has accumulated on the relationship between the stability of protein structure and its function; we may be able to fine-tune the activity of biological catalysts and create completely synthetic analogs of them. Recently, work appeared that reported the cloning of the first synthetic enzyme gene encoding the active fragment of the ribonuclease molecule.

III. Applications of Protein Engineering

Currently, the most popular application of protein engineering is to modify the catalytic properties of enzymes to develop “environmentally friendly” industrial processes. From an environmental point of view, enzymes are the most acceptable of all catalysts used in industry. This is ensured by the ability of biocatalysts to dissolve in water and fully function in an environment with a neutral pH and at relatively low temperatures. In addition, due to their high specificity, the use of biocatalysts results in very few unwanted production by-products. Environmentally friendly and energy-saving industrial processes using biocatalysts have long been actively introduced in the chemical, textile, pharmaceutical, pulp and paper, food, energy and other areas of modern industry.

However, some characteristics of biocatalysts make their use unacceptable in some cases. For example, most enzymes break down when the temperature increases. Scientists are trying to overcome such obstacles and increase the stability of enzymes under harsh production conditions using protein engineering techniques.

In addition to industrial applications, protein engineering has found a worthy place in medical developments. Researchers synthesize proteins that can bind to and neutralize viruses and mutant genes that cause tumors; creating highly effective vaccines and studying cell surface receptor proteins, which are often targets for pharmaceuticals. Food scientists use protein engineering to improve the preservation properties of plant-based proteins and gelling agents or thickening agents.

3.1 Peptide and epitope libraries

Libraries of peptides immobilized on microcarriers have a significant drawback: they require the use of purified receptors in soluble form during screening. At the same time, in most cases, membrane-associated receptors are most often used in biological tests carried out for basic and pharmacological research. According to the second method, peptide libraries are obtained using solid-phase peptide synthesis, in which at each stage of the chemical addition of the next amino acid to the growing peptide chains, equimolar mixtures of all or some precursor amino acids are used. At the final stage of synthesis, the peptides are separated from the carrier, i.e. converting them into soluble form. The third approach to constructing peptide libraries, which we are now describing, became feasible precisely thanks to the development of genetic engineering methods. It perfectly illustrates the capabilities of such methods and is undoubtedly a major advance in their application. In this regard, we will consider in more detail the results of using peptide libraries in the study of epitopes (antigenic determinants) of proteins.

3.2 Reporter proteins in fusion proteins

In another case, fusion proteins are used to obtain high levels of expression of short peptides in bacterial cells due to the stabilization of these peptides within the fusion proteins. Hybrid proteins are often used to identify and purify difficult-to-detect recombinant proteins. For example, by attaching galactosidase to the C-terminus of the protein under study as a reporter protein, it is possible to purify the recombinant protein based on galactosidase activity, determining its antigenic determinants by immunochemical methods. By combining DNA fragments containing open reading frames (ORFs) with the genes of reporter proteins, it is possible to purify such hybrid proteins based on the activity of the reporter protein and use them to immunize laboratory animals. The resulting antibodies are then used to purify the native protein, which includes the recombinant polypeptide encoded by the ORF, and thereby identify the cloned gene fragment.

With the help of hybrid proteins, the inverse problem of cloning an unknown gene, to the protein product of which there are antibodies, is also solved. In this case, a clone library of nucleotide sequences representing ORFs of unknown genes is constructed in vectors that allow the ORF to be cloned to be connected in the same reading frame with the reporter gene. The hybrid proteins formed as a result of the expression of these recombinant genes are identified using antibodies using enzyme immunoassay methods. Hybrid genes combining secreted proteins and reporter proteins make it possible to study in new ways the mechanisms of secretion, as well as the localization and movement of secreted proteins in tissues.

3.3 Some achievements of protein engineering

By replacing several amino acid residues of bacteriophage T4 lysozyme with cysteine, an enzyme with a large number of disulfide bonds was obtained, due to which this enzyme retained its activity at higher temperatures.

Replacing a cysteine residue with a serine residue in the molecule of human β-interferon, synthesized by Escherichia coli, prevented the formation of intermolecular complexes, which reduced the antiviral activity of this drug by approximately 10 times.

Replacing the threonine residue with a proline residue in the molecule of the enzyme tyrosyl-tRNA synthetase increased the catalytic activity of this enzyme tenfold: it began to quickly attach tyrosine to the tRNA that transfers this amino acid to the ribosome during translation.

Subtilisins are serine-rich enzymes that break down proteins. They are secreted by many bacteria and are widely used by humans for biodegradation. They firmly bind calcium atoms, increasing their stability. However, in industrial processes there are chemical compounds that bind calcium, after which subtilisins lose their activity. By changing the gene, the scientists removed amino acids from the enzyme that are involved in calcium binding and replaced one amino acid with another in order to increase the stability of subtilisin. The modified enzyme turned out to be stable and functionally active under conditions close to industrial ones.

The possibility of creating an enzyme that functions like a restriction enzyme that cleaves DNA in strictly defined places was shown. Scientists created a hybrid protein, one fragment of which recognized a specific sequence of nucleotide residues in a DNA molecule, and the other fragmented DNA in this region.

Tissue plasminogen activator is an enzyme that is used clinically to dissolve blood clots. Unfortunately, it is quickly eliminated from the circulatory system and must be administered repeatedly or in large doses, which leads to side effects. By introducing three targeted mutations into the gene of this enzyme, we obtained a long-lived enzyme with increased affinity for degraded fibrin and with the same fibrinolytic activity as the original enzyme.

By replacing one amino acid in the insulin molecule, scientists ensured that when this hormone was administered subcutaneously to patients with diabetes, the change in the concentration of this hormone in the blood was close to the physiological one that occurs after eating.

There are three classes of interferons that have antiviral and anticancer activity, but exhibit different specificities. It was tempting to create a hybrid interferon that would have the properties of the three types of interferons. Hybrid genes were created that included fragments of natural interferon genes of several types. Some of these genes, being integrated into bacterial cells, ensured the synthesis of hybrid interferons with greater anticancer activity than the parent molecules.

Natural human growth hormone binds not only to the receptor of this hormone, but also to the receptor of another hormone - prolactin. In order to avoid unwanted side effects during treatment, scientists decided to eliminate the possibility of growth hormone attaching to the prolactin receptor. They achieved this by replacing some amino acids in the primary structure of growth hormone using genetic engineering.

While developing drugs against HIV infection, scientists obtained a hybrid protein, one fragment of which ensured the specific binding of this protein only to lymphocytes affected by the virus, another fragment ensured the penetration of the hybrid protein into the affected cell, and another fragment disrupted protein synthesis in the affected cell, which led to her death.

Proteins are the main targets for drugs. Currently, about 500 targets for drug action are known. In the coming years, their number will increase to 10,000, which will make it possible to create new, more effective and safe drugs. Recently, fundamentally new approaches to drug discovery have been developed: not single proteins, but their complexes, protein-protein interactions and protein folding are considered as targets.

Conclusion

Protein engineering technology is used (often in combination with the recombinant DNA method) to improve the properties of existing proteins (enzymes, antibodies, cellular receptors) and create new proteins that do not exist in nature. Such proteins are used to create medicines, in food processing and in industrial production.

protein engineering mutagenesis modified

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25.Protein engineering. // Wikipedia, the free encyclopedia.

Genetic engineering is the in vitro construction of functionally active genetic structures (recombinant DNA), or in other words, the creation of artificial genetic programs (Baev A.A.). According to E.S. Piruzyan genetic engineering is a system of experimental techniques that makes it possible to construct artificial genetic structures in the laboratory (in vitro) in the form of so-called recombinant or hybrid DNA molecules.

Genetic engineering is the production of new combinations of genetic material by manipulation of nucleic acid molecules outside the cell and the transfer of the created gene constructs into a living organism, as a result of which their inclusion and activity in this organism and its offspring is achieved. We are talking about the directed, according to a predetermined program, construction of molecular genetic systems outside the body with their subsequent introduction into a living organism. In this case, recombinant DNA becomes an integral part of the genetic apparatus of the recipient organism and imparts to it new unique genetic, biochemical, and then physiological properties.

The goal of applied genetic engineering is to design such recombinant DNA molecules that, when introduced into the genetic apparatus, would give the body properties useful to humans. For example, the production of “biological reactors” - microorganisms, plants and animals that produce substances that are pharmacologically significant for humans, the creation of plant varieties and animal breeds with certain traits valuable to humans. Genetic engineering methods make it possible to carry out genetic certification, diagnose genetic diseases, create DNA vaccines, and conduct gene therapy for various diseases.

Recombinant DNA technology uses the following methods:

Specific cleavage of DNA by restriction nucleases, accelerating the isolation and manipulation of individual genes;

Rapid sequencing of all nucleotides in a purified DNA fragment, which makes it possible to determine the boundaries of the gene and the amino acid sequence encoded by it;

Construction of recombinant DNA;

Nucleic acid hybridization, which allows the detection of specific RNA or DNA sequences with greater accuracy and sensitivity, based on their ability to bind complementary nucleic acid sequences;

DNA cloning: in vitro amplification using a polymerase chain reaction or introduction of a DNA fragment into a bacterial cell, which, after such transformation, reproduces this fragment in millions of copies;

Introduction of recombinant DNA into cells or organisms.

The construction of recombinant molecules is carried out using a number of enzymes - primarily restriction enzymes. Currently, over 400 different restriction enzymes are used. These enzymes are synthesized by a wide variety of microorganisms.

Restriction enzymes recognize and cleave specific nucleotide sequences in a double-stranded DNA molecule. However, restriction enzymes alone are not sufficient for molecular cloning because the hydrogen bonds between the four bases that form the sticky ends are not strong enough to hold two DNA fragments together.

One part of the recombinant DNA molecule carries the desired gene that is supposed to be cloned, the other contains the information necessary for the replication of recombinant DNA in the cell.

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480 rub. | 150 UAH | $7.5 ", MOUSEOFF, FGCOLOR, "#FFFFCC",BGCOLOR, "#393939");" onMouseOut="return nd();"> Dissertation - 480 RUR, delivery 10 minutes, around the clock, seven days a week and holidays

Efimov Grigory Alexandrovich. New genetically engineered proteins based on recombinant antibodies against TNF: dissertation... Candidate of Biological Sciences: 01/03/03 / Efimov Grigory Aleksandrovich; [Place of defense: V.A. Engelhard Institute of Molecular Biology RAS]. - Moscow, 2015. - 122 s.

Introduction

Literature review 9

1. History of the discovery of tnf 9

2. TNF 10 superfamily

3. System structure tnfnfr 12

4. Functions tnf 15

5. The role of TNF in the pathogenesis of rheumatoid arthritis and other autoimmune diseases 16

6. Therapeutic blocking tnf 18

7. Side effects and limitations of antinf therapy 23

8. New approaches and prospects for tnf blocking 25

Materials and research methods 29

1. Production and characterization of a new camel single domain antibody to human tnf 29

Expression and purification of single domain antibody Vhh41 29

Evaluation of binding of the Vhh41 antibody to human TNF by ELISA 30

Study of the interaction between Vhh41 and human TNF using surface plasma resonance method 31

Studies on the ability of Vhh41 to block human TNF 31

2. Design, preparation and characterization of hybrid proteins of TNF fluorescent sensors 32

Construction of genes encoding TNF sensors. 32

Expression and purification of TNF fluorescent sensors. 33

Analysis of the interaction of Vhh41-K with recombinant TNF. 34

Study of the biological properties of the Vhh41-KTNFin fluorescent sensor in vitro and in vivo. Z5

Study of the ability of a fluorescent sensor to bind TNF in vivo 36

Intravital study of TNF expression using the obtained fluorescent sensor...39

3. Preparation and characterization of single-chain ANTINF antibody 40

Study of mouse monoclonal antibody F10 40

Construction and expression of single chain antibody ahT-4 41

Measurement of biological activity of single chain antibody ahT-4 42

4. Preparation and characterization of chimeric antinf antibody 43

5. Construction, production and characterization of bispecific antibodies A9 and MA9 43

Construction, expression and purification of antibodies A9 and tA9 43 Interaction of antibodies A9 and tA9 with recombinant human TNF using the method

surface plasma resonance 44

Cytotoxic test 45

Cytofluorimetry 45

Evaluation of the ability of bispecific antibody A9 to retain human TNF at

surface of macrophages 45

6. Comparative assessment of the effectiveness of systemic and selective blocking of macrophage TNF 46

Model of acute hepatotoxicity induced by administration of JIIJC/D-galactosamine 46

Results and discussion 48

1. Production and characterization of a new recombinant single domain antibody that specifically binds to human TNF, but does not block its biological activity 50

Creation of a genetic construct encoding a recombinant single-domain antibody

Expression and purification of recombinant single domain antibody Vhh41 52

Analysis of the interaction of single-domain antibody Vhh41 with human TNF 53

Analysis of the ability of the Vhh41 antibody to block the biological activity of human TNF.54

2. Design, production and characterization of TNF molecular sensors for intravital study of tnf expression based on single domain recombinant antibodies and red fluorescent protein 56

Preparation of genetic constructs encoding the TNF fluorescent sensor Vhh41-Ku

control fusion proteins 56

Expression and purification of the TNF fluorescent sensor Vhh41-K. 57

Analysis of the interaction of the TNF fluorescent sensor Vhh41-K with recombinant mouse TNF

and person 58

Study of the biological properties of the fluorescent sensor TNF Vhh41-Kin in vitro and in vivo. 61

Study of the ability of a fluorescent sensor to bind TNF in vivo 66

Intravital study of TNF expression using the obtained fluorescent sensor... 69

3. Preparation and characterization of a recombinant single-chain antibody that blocks the biological activity of TNF 72

Measurement of activity of mouse monoclonal antibody F10 72

Construction of a single-chain antibody based on variable fragments of lung and

heavy chains of mouse monoclonal antibody F 10 74

Measurement of single chain antibody activity ahT-4 75

4. Development and analysis of a chimeric antibody against human TNF

Comparison of the kinetics of interactions of the chimeric antibody 13239 and infliximab with

recombinant human TNF 77 Comparison of the neutralizing activity of the chimeric antibody 13239 with the activity

infliximab in vitro 79

In vivo activity assay of chimeric antibody 13239 80

5. Design, production and characterization of a selective tnf blocker produced by cells of the monocyte-macrophage series 82

Molecular cloning, expression and purification of bispecific antibodies 82

Interaction of antibodies A9 and tA9 with recombinant human TNF 86

Blocking of TNF-dependent cytotoxicity in vitro by antibodies A9 and tA9 87

Analysis of the binding of antibodies A9 and tA9 to the surface of macrophages through interaction with

surface molecule F4/80 89

Retention of endogenously produced human TNF on the surface of macrophages

bispecific antibody A9 93

6. Physiologically significant selective blocking of tnf produced by cells of the monocyte-macrophage lineage in vivo 96

Comparative assessment of the effectiveness of targeted blocking of TNF produced by cells of the monocyte-macrophage lineage and systemic blocking of TNF in a model of acute

hepatotoxicity 96

Conclusion 99

References 100

The role of TNF in the pathogenesis of rheumatoid arthritis and other autoimmune diseases

The first experience with anti-cytokine therapy was carried out in 1985, when polyclonal anti-NF rabbit serum was administered to mice, which prevented the development of lethal hepatotoxicity induced by LPS administration. Similar results were obtained in monkeys: baboons treated with a mouse monoclonal antibody against human TNF survived intravenous injection of a lethal dose of E. coli [104].

The first therapeutic TNF blocker was developed based on the high-affinity murine monoclonal antibody A2, derived from mice immunized with human TNF. Because antibodies of other types have significant differences in amino acid sequence, they are unsuitable for long-term therapeutic use in humans. Therefore, using genetic engineering, the mouse constant domains of the heavy and light chains were replaced with human ones. The variable regions that bind the antigen remained unchanged. Such antibodies are called chimeric. Subsequently, this first therapeutic antibody against TNF received the international nonproprietary name - infliximab.

One of the most obvious areas of application for antiNF therapy has been in the treatment of sepsis. However, clinical studies have not shown significant results, which is apparently due to the fact that by the time the clinical picture of sepsis develops, irreversible signaling cascades have already been launched.

By this time, many facts had already accumulated indicating the participation of TNF in the pathogenesis of rheumatoid arthritis, so this disease was chosen as the next potential target for antiNF therapy. Pilot studies of infliximab in rheumatoid arthritis showed promising results, and a further randomized, double-blind study confirmed the effectiveness of antiNF therapy in the treatment of autoimmune diseases. However, after repeated injections, some patients developed antibodies specific to mouse amino acid sequences in the variable domains, which reduced the effectiveness of therapy.

A double-blind, randomized study showed that infliximab has a synergistic effect with low-dose methotrexate, a cytostatic drug used for monotherapy of RA. When combined, these two drugs are more effective and the immunogenicity of infliximab is reduced. Subsequent phase II/III clinical trials led to the approval of infliximab for the treatment of RA.

The mechanism of action of infliximab is mainly due to the binding of soluble TNF in the systemic circulation and in places of local overexpression (synovial cavity in RA). But, in addition, infliximab is able to bind to the transmembrane form of TNF and cause lysis of cells carrying it on their surface through the mechanism of antibody-dependent cytotoxicity.

AntiNF therapy breaks the pathological signaling cascade and leads to a decrease in the inflammatory response, but in addition, it is able to balance the dysregulated immune system. With the introduction of TNF inhibitors, the balance of T-effector and T-regulatory cells shifts.

AntiNF therapy is not etiotropic therapy and theoretically should be used throughout the patient’s life, however, in some cases it is possible to achieve stable remission, which persists even after discontinuation of antiNF therapy.

TNF blockers have also shown their effectiveness in the treatment of other autoimmune and inflammatory diseases: TNF has been shown to play a significant role in the pathogenesis of Crohn's disease - it is overexpressed in inflamed areas of the intestine. Preliminary successes in treating Crohn's disease resistant to standard therapy with infliximab were later confirmed in randomized clinical trials, resulting in infliximab being approved for the treatment of this disease as well.

The pathogenesis of ankylosing spondylitis (ankylosing spondylitis), another chronic systemic autoimmune disease primarily affecting the joints, is also due to overexpression of TNF. Clinical trials of infliximab have been successful for this disease as well. In addition, antiNF therapy has shown high effectiveness in the treatment of psoriasis and psoriatic arthritis.

To date, infliximab and other TNF blockers are approved as therapeutic agents for the following autoimmune diseases: rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis. In addition, TNF antagonists have shown positive results in the treatment of sarcoidosis, Wegener's granulomatosis, Behçet's disease and other chronic diseases.

Indications that TNF plays a role in the pathogenesis of multiple sclerosis have been confirmed by experiments in laboratory animals. Administration of TNF increased the symptoms of experimental autoimmune encephalomyelitis in rats, and administration of antiNF antibodies prevented the development of this disease.

However, clinical trials for the treatment of multiple sclerosis with infliximab and another TNF blocker, lenercept (soluble TNFR1), did not produce a significant clinical response. Moreover, in some patients

A model autoimmune disease, the pathogenesis of which is similar to the pathogenesis of multiple sclerosis. There was an increase in the clinical symptoms of the disease and an increase in cellularity and the level of immunoglobulins in the cerebrospinal fluid, and an increase in the number of lesions on magnetic resonance imaging.

The success of infliximab has given impetus to the development of new molecules capable of blocking signal transmission through TNFR. In addition, mouse sequences in the variable domains of the heavy and light chains of infliximab caused the production of secondary antibodies in some patients, which blocked the effect of infliximab and made the patients refractory to therapy. To overcome this limitation, a route was taken to create inhibitors with fully human amino acid sequences.

To date, in addition to infliximab, four TNF antagonists have been approved for clinical use (see Figure 2):

Etanercept is a recombinant TNF inhibitor designed from soluble TNFR2. Its development was based on data that a soluble form of the second TNF receptor is present in the human body. TNFR2, “exfoliated” by metalloproteases, is an additional link in the regulation of TNF activity. Etanercept is a dimer of the extracellular portion of TNFR2 genetically fused to the Fc portion of immunoglobulin IgG1. Connection to the antibody constant region significantly increases the half-life of the drug in the systemic circulation due to protein recycling through the FcRn receptor. The neutralizing activity of the fusion protein was demonstrated in both in vitro and in vivo experiments, and was later confirmed in clinical trials in patients suffering from rheumatoid arthritis.

However, in the treatment of inflammatory bowel diseases, etanercept, unlike infliximab, has not shown therapeutic efficacy. The experimental TNF blocker onercept, derived from another TNF receptor, TNFR1 (p55), despite encouraging pilot clinical studies in a randomized, placebo-controlled, double-blind study, also did not show effectiveness in the treatment of Crohn's disease. An in vitro study examining T lymphocytes from the lamina propria of patients with Crohn's disease showed that while both infliximab and etanercept blocked TNF, only infliximab bound to T cells at the lesion and induced apoptosis in them. This may explain the difference in effectiveness of antibody-based and recombinant receptor blockers in inflammatory bowel diseases.

Study of the interaction between Vhh41 and human TNF using surface plasma resonance

The genetic construct encoding the bispecific antibody A9 was assembled by HSH by a reaction with 4 primers similar to that described above for the single-chain antibody gene ahT-4. The resulting sequence consisted of: a single-domain antiNF antibody gene, then a sequence encoding a linker species (Gly4Ser)3, and a single-chain anti-P4/80 antibody gene (kindly provided by S. Gordon and M. Stacey). The recognition sites for restriction enzymes Ncol and Xhol were included in the sequence of the forward and reverse primers, respectively. After restriction of the PCR product and cloning it into the expression vector pET-28b (Novagen), the sequence encoding the polyhexidine tag was found at the 3rd end in the same reading frame. To obtain the control antibody wA9, the mutant anti-G4/80 scFv gene containing glycine-serine inserts instead of CDR sequences was synthesized de-novo (Geneart, Germany) and cloned instead of the native anti-F4/80 gene (see Fig. 31B).

Expression vectors carrying inserts encoding A9 and mA9 were used to transform E. coli cells of the Rosetta2(DE3)pLysS strain (Novagen). The best producing clones were selected by colony immunoblotting using nickel-conjugated peroxidase (Pierce, 15165). Bacterial cultures were grown in LB medium containing 50 cg/ml carbenicillin (Sigma-C1389) and 50 cg/ml chloramphenicol (Sigma-C1863) to logarithmic phase, and then expression was induced by 0.2 mM IPTG. After 4 hours, the cultures were centrifuged at 3200 g for 30 minutes. The pellets were frozen and then resuspended in lysis buffer (50 mM TrisHCl, 300 MMNaCl, 5% glycerol, 0.5% Triton X-100 detergent, 10,000 U/ml lysozyme, 10 mM P-mercaptoethanol) and then disrupted using an ultrasonic homogenizer. The lysates were centrifuged at 17,000 g for 40 min, the supernatants were collected and filtered through a filter with a pore diameter of 0.22 cm. Bispecific antibodies A9 and tA9 were purified from cleared supernatants on a chromatographic column containing agarose conjugated to Ni-nitriloacetic acid (Invitrogen R90115). Affinity chromatography was performed according to the manufacturer's protocol. The resulting eluate was concentrated, dialyzed against phosphate-buffered saline, followed by filtration through a 0.22 μm filter. The protein concentration in the solution was measured using a reaction with 2,2-bicinchoninic acid (PIERCE 23225 kit) according to the manufacturer's protocol. The homogeneity of the resulting preparation was tested by electrophoresis in a 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, followed by Coomassie staining.

Interaction of antibodies A9 and tA9 with recombinant human TNF using surface plasmon resonance.

A comparison of the affinities and kinetics of interaction of antibodies A9 and mA9 with recombinant human TNF was carried out on a ProteOn XPR36 instrument (Bio-Rad). During the measurement of all interactions, phosphate-buffered saline having a pH of 7.4 was used, to which the detergent Tween 20 was added to a concentration of 0.005%, the chip surface temperature was 25 C. Recombinant human TNF was expressed in E. coli according to the previously described method . Antibodies A9 and tA9 at a concentration of 50 nM were immobilized through the amino group on the surface of a biochip with a modified alginate polymer surface (Bio-Rad 176-5011). Then the analyte (human TNF) in five twofold decreasing concentrations (50 -3 nM) was applied into five parallel channels. A buffer containing no antibody was introduced into the sixth channel for normalization. The analysis of the obtained sensorgrams was carried out in the ProteOn Manager program (Bio-Rad) using the Langmuir model.

In experiments with peritoneal macrophages, peritoneal cells were isolated from wild-type (C57BL/6) mice and immediately stained using fluorochrome-conjugated antibodies. To obtain bone marrow macrophages, bone marrow was isolated, after which the cells were cultured for 10 days in conditioned medium (obtained on the L929 line), then the cells were removed from the plastic with ice-cold phosphate buffer.

Before staining, the Fc-gamma receptor was blocked, then the cells were incubated with A9 or tA9 antibodies or buffer, after which the cells were washed and stained in one of three ways: 1) polyclonal rabbit antibodies to hTNF-VnH, then with secondary antibodies to rabbit IgG conjugated to fluorochrome. 2) monoclonal mouse antibodies to the hexahistidine sequence (Novagen - 70796), then with secondary antibodies to mouse IgG conjugated to a fluorochrome. 3) recombinant human TNF was added to the cells, and then fluorochrome-labeled monoclonal antiNF antibodies (Miltenyi Biotec - clone: cA2).

In addition, the cells were stained with anti-P4/80 and anti-CD 1 lb antibodies conjugated to fluorochromes. Samples were analyzed on either an F ACS Aria (BDBiosciences) or a Guava EasyCyte 8HT (Millipore) and the resulting data were processed using FlowJo software (Treestar Inc.).

Evaluation of the ability of bispecific antibody A9 to retain human TNF on the surface of macrophages.

Peritoneal macrophages from human TNF-producing mice were isolated and seeded at 100 thousand cells per well in 96-well culture plates. The cells were incubated for 2 hours at 37C, 5% CO2, after which the unattached cells were washed off with warm phosphate buffer. Then the cells were incubated overnight at 37C, 5% CO2. After washing with 200 μl of warm DMEM, the cells were incubated with A9 antibodies at a concentration of 2 μg/ml or with DMEM for 30 minutes at 37C. After another wash, cells were stimulated with LPS (Sigma, L2630) at a concentration of 100 ng/ml. After 4 h, culture supernatants were collected and human TNF concentration was measured using an ELISA kit (eBioscience, 88-7346) according to the manufacturer's protocol.

Bone marrow from human TNF-producing mice was isolated, after which the cells were cultured for 10 days in conditioned medium (obtained on the L929 line), then the cells were removed from the plastic with ice-cold phosphate buffer. The number of living cells was counted and they were seeded into 96-well plates at a concentration of 50,000 cells/well. Cells were then supplemented with 250 mM of A9 antibody or hTNF-VffH single-domain antibody or empty medium (DMEM). Cells were incubated with antibodies for 30 min. The wells were then washed with phosphate-buffered saline. After this, TNF production was stimulated by LPS (Sigma - L2630) at a concentration of 100 ng/ml. After 4 hours, the supernatants were collected, and the concentration of TNF in them was measured using a cytotoxic test on the L929 mouse fibrosarcoma line using a protocol similar to that described above.

Study of the biological properties of the fluorescent sensor Vhh41-KTNFin vitro and in vivo

Based on experimental data obtained from mouse strains in which the Tnf gene has been deleted in separate cell populations, a hypothesis has been formulated regarding the possible different functions of TNF produced by different types of immunocytes. Thus, it was recently shown that in a model of experimental tuberculosis infection, TNF produced by T lymphocytes, but not myeloid cells, has a unique protective function. In addition, our laboratory has obtained data indicating the pathogenic properties of TNF from myeloid cells in autoimmune diseases. The therapeutically applied complete blocking of PMB does not take these features into account. As part of the development of this hypothesis, specific inhibition of TNF produced by cells of the monocyte-macrophage line was chosen, which could have a significant advantage over systemic blocking of this cytokine. In particular, an intact signal from TNF produced by B and T lymphocytes could reduce the incidence of side effects, and, in addition, make anti-NF therapy effective in those diseases for which TNF blockers have previously shown no clinical effectiveness, or even caused increased symptoms. In addition, this approach can potentially reduce the required dose due to targeted delivery to producer cells.

To test this assumption, we constructed and tested a bispecific antibody, which with one part binds to the surface of macrophages due to interaction with the transmembrane molecule F4/80, and with the second specificity captures and blocks the TNF they produce.

Molecular cloning, expression and purification of bispecific antibodies. The bispecific antibody, a selective blocker of macrophage TNF, was named A9. To create the genetic construct encoding it, a single-domain anti-NF blocking antibody hTNF-VffH and a single-chain antibody (scFv) against the macrophage surface marker F4/80 were used (kindly provided by S. Gordon (University of Oxford, UK) and M. Stacey (University of Leeds, UK) The sequences encoding both antibodies were amplified using polymerase chain reaction (PCR) and cloned into an expression vector so that they were in the same reading frame, and between them a nucleotide sequence encoding a flexible glycine-serine linker (GSGGGGSG) was formed. At the C-terminus of the sequence there is a sequence encoding a histidine hexamer for subsequent protein purification (Fig. 31).

Design of the bispecific antibody A9, a schematic representation of its mechanism of action, the structure of genetic constructs encoding the bispecific antibody A9 and the control systemic TNF blocker antibody tA9. (A) Bispecific antibody A9 consists of a single-domain antibody (VHH) against human TNF and a single-chain antibody (scFv) against the surface molecule F4/80 expressed on monocytes and macrophages. (B) Principle of selective blocking of TNF produced by macrophages: A9 binds to the surface of macrophages and captures TNF released from their surface, preventing it from entering the systemic circulation. (B) Scheme of the genetic design of the bispecific antibody A9 and the control systemic TNF blocker tA9. The single-domain antiNF antibody gene is followed by a sequence encoding a flexible glycine-serine linker and then a single-chain anti-F4/80 antibody gene. This is followed by a sequence encoding the histidine hexamer for affinity purification. The control antibody tA9 has a similar sequence, except that 6 hypervariable regions of the anti-P4/80 antibody are replaced with sequences of the type (Gly3Ser)n, which prevents the antibody from binding to the surface of macrophages and turns it into a systemic TNF inhibitor.

To study the effects of specific blocking of TNF produced by macrophages, a control systemic blocker was needed. To avoid effects associated with differences in antibody affinity, it was decided to use a blocker with a similar A9 TNF-binding site. And in order to exclude the influence of other factors, in particular the isoelectric point and molecular weight, which can affect the half-life, the control antibody should be as close as possible in the primary amino acid sequence to the one being studied. Therefore, we constructed a control antibody - tA9, which has the same structure and amino acid sequence as A9, except that 6 of its hypervariable regions in the anti-P4/80 scFv were replaced with sequences of the form (Gly3Ser)n, the same length as original CDR regions (see Fig. 31 B).

Both antibodies were expressed in a bacterial system and purified by affinity chromatography.

The size of antibody A9, determined by electrophoretic mobility and HPLC data, corresponded to the calculated molecular mass of 45 kDa (Fig. 32). chromatography. On the left are the molecular weights of proteins. (B) Chromatogram of bispecific antibody A9 (in red) superimposed on a chromatogram of molecular weight markers. (B) Function of molecule transit time as a function of molecular weight. The calculated molecular weight of the bispecific antibody A9 was 43.4 kDa.

Interaction of antibodies A9 and tA9 with recombinant human TNF. The kinetics of interaction of antibodies A9 and tA9 with recombinant human TNF was measured by surface plasmon resonance. To do this, both antibodies at a concentration of 50 nM were immobilized on the surface of the sensor chip, after which recombinant human TNF in serial dilutions of 50-3 nM was applied as an analyte, and the interaction kinetics were measured on a ProteOn XPR36 device. Both antibodies showed high affinity: Kd of A9 and tA9 was 85 and 95 pM, respectively. This confirms that the introduced mutations did not affect TNF binding. In addition, both antibodies had similar parameters of binding rate (Kforward, on-rate) and dissociation rate (Kreverse, off-rate) - shown in Fig. 33 and in Tab. 3. The slow dissociation rate should allow antibody A9 to retain bound TNF.

Production and characterization of a new recombinant single domain antibody that specifically binds to human TNF but does not block its biological activity

The kinetics of interaction of antibodies A9 and tA9 with recombinant human TNF was measured by surface plasmon resonance. To do this, both antibodies at a concentration of 50 nM were immobilized on the surface of the sensor chip, after which recombinant human TNF in serial dilutions of 50-3 nM was applied as an analyte, and the interaction kinetics were measured on a ProteOn XPR36 device. Both antibodies showed high affinity: Kd of A9 and tA9 was 85 and 95 pM, respectively. This confirms that the introduced mutations did not affect TNF binding. In addition, both antibodies had similar parameters of binding rate (Kforward, on-rate) and dissociation rate (Kreverse, off-rate) - shown in Fig. 33 and in Tab. 3. The slow dissociation rate should allow antibody A9 to retain bound TNF.

Kinetics of interaction of bispecific antibody A9 and control antibody tA9 with recombinant human TNF. (A) Interaction curves (sensograms) of recombinant human TNF at concentrations of 50 nM - 3 nM with a sensor chip on which the bispecific antibody A9 and the control antibody tA9 were immobilized are shown. The abscissa axis shows time in seconds, and the ordinate axis shows the resonance angle shift in conventional units (CU). (B) For each group of sensorograms, the values of the binding rate (op-rate), dissociation rate (off-rate) and dissociation constant (Kd) were calculated. The resulting average values, as well as the standard deviation (SD), are plotted on the isoaffinity diagram. Diagonal lines correspond to the indicated dissociation constant values.

To evaluate the comparative activity of antibody A9 in inhibiting the biological effects of TNF, cytotoxic testing was performed on the L929 murine fibrosarcoma line. Serial dilutions of A9 and tA9 antibodies were added to constant concentrations of recombinant human TNF and actinomycin-D. According to the data obtained, antibodies A9 and tA9 have similar antiNF activity (Fig. 34 A). In addition, it was confirmed that the activity of the bispecific antibody A9 corresponds to the activity of the single-domain antiNF antibody hTNF-VffH, which is part of A9 and tA9 (Fig. 34 B). 10 10 10

AntiNF activity of the bispecific antibody A9, the control antibody mA9, and the single domain antibody hTNF-VffH. (A) Comparison of activity of bispecific antibody A9 and control antibody tA9. The survival curve of murine fibrosarcoma L929 cells under simultaneous exposure to a constant dose of human TNF and decreasing doses of antibodies A9 and mA9 is presented. (B) Comparison of the activity of the bispecific antibody A9 and the single-domain antibody hTNF-VnH. The survival curve of murine fibrosarcoma L929 cells is presented under simultaneous exposure to a constant dose of human TNF and decreasing doses of antibodies A9 and hTNF-VHH. The comparison was carried out in molar concentrations in order to exclude the influence of differences in molar mass on the determined activity of the antibody.

Analysis of the binding of antibodies A9 and tA9 to the surface of macrophages through interaction with the surface molecule F4/80.

The ability of the bispecific antibody A9 to specifically bind to the surface of macrophages was assessed by flow cytometry. To do this, cells isolated from the peritoneal cavity were incubated with A9 antibodies, after which they were stained for macrophage markers CD1 lb and F4/80, while specific staining was carried out for the bispecific A9 antibody through antibodies to VHH OR antibodies to the polyhistidine tag. The samples were then subjected to flow cytometry and analysis.

These experiments showed that bispecific antibody A9 is able to bind to the surface of peritoneal cells expressing F4/80 and CD1 lb on their surface (monocytes and macrophages) (Fig. 35 A - D). At the same time, A9 does not bind to cells of the peritoneal cavity that do not have these markers (mainly lymphocytes) (Fig. 35 E and F). A decrease in the level of parallel anti-F4/80 staining upon addition of the A9 antibody due to competition between two antibodies for binding to the target confirms that A9 specifically interacts with this molecule on the cell surface (Fig. 35 G and 3).

The name Mas-1 is also used. A component of the receptor for the S3 component of the complement system. In mice it is expressed on monocytes, macrophages and microglial cells. bispecific antibody

Peritoneal cavity cells were incubated with or without bispecific antibody A9 (shown in red) and then stained with fluorescently labeled antibodies to surface markers specific for monocyte-macrophage cells and with antibodies specific to A9. Then the obtained samples were analyzed by flow cytometry. (A, B, E, G) staining with antibodies to the VHH domain. (B, D, E, 3) staining with antibodies to the polyhexidine sequence. (A, B) bispecific antibody A9 binds to cells selected for high levels of expression of F4/80 and CD1 lb (macrophages). In the histogram shown, the horizontal axis shows the fluorescence value in the staining channel on A9, and the vertical axis shows the normalized frequency of occurrence of the event. (C, D) the same in the form of a scatter histogram. The horizontal axis shows the fluorescence value in the staining channel on A9, and the vertical axis shows the fluorescence value in the staining channel on F4/80. (D, F) -bispecific antibody A9 does not bind to cells of the peritoneal cavity that do not express F4/80 and CD1 lb (lymphocytes). In the histogram shown, the horizontal axis shows the fluorescence value in the staining channel on A9, and the vertical axis shows the normalized frequency of occurrence of the event. (G, 3) -incubation with bispecific antibody A9 reduces the intensity of staining for F4/80. In the histogram shown, the horizontal axis shows the fluorescence value in the staining channel at F4/80, and the vertical axis shows the normalized frequency of occurrence of the event.

Bone marrow macrophages were incubated with bispecific A9 antibody (shown in red), without it (shown in blue), or with control tA9 antibody (shown in black) and then stained with A9/tA9-specific antibodies. The obtained samples were analyzed by flow cytometry. (A) Bispecific antibody A9 binds specifically to bone marrow macrophages. In the histogram shown, the horizontal axis shows the fluorescence value in the staining channel on A9, and the vertical axis shows the normalized frequency of occurrence of the event. (B) control antibody wA9 fails to bind to bone marrow macrophages. In the histogram shown, the horizontal axis shows the fluorescence value in the staining channel on wA9, and the vertical axis shows the normalized frequency of occurrence of the event.

In addition, additional cytofluorometric experiments showed that the A9 antibody, when attached to the surface of macrophages, is able to simultaneously bind exogenously added human TNF (Fig. 37). This confirms that both subunits of the bispecific antibody are functionally active at the same time, and that the binding of two antigens at the same time is sterically possible.

Peritoneal cavity cells were incubated with or without bispecific antibody A9 (shown in red), then with recombinant human TNF, and then stained with fluorescently labeled antibodies to surface markers specific for monocyte-macrophage cells, as well as antibodies specific for human TNF. The obtained samples were analyzed by flow cytometry. (A) bispecific antibody A9 is capable of retaining human TNF on the surface of macrophages (cells selected for high levels of F4/80 and CD1 lb expression). In the histogram shown, the horizontal axis shows the fluorescence value in the TNF staining channel, and the vertical axis shows the normalized frequency of occurrence of the event. (B) The same data in scatter histogram form. The horizontal axis shows the fluorescence values in the TNF staining channel, and the vertical axis shows the fluorescence values in the F4/80 staining channel.

PROTEIN ENGINEERING, a branch of molecular biology and bioengineering, the tasks of which include targeted changes in the structure of natural proteins and the production of new proteins with specified properties. Protein engineering arose in the early 1980s, when genetic engineering methods were developed that made it possible to obtain various natural proteins using bacteria or yeast, as well as in a certain way to change the structure of genes and, accordingly, the amino acid sequence (primary structure) of the proteins they encode. Based on the principles of organization of protein molecules, the relationship between the structure and function of proteins, protein engineering creates a scientifically based technology for targeted changes in their structure. With the help of protein engineering, it is possible to increase the thermal stability of proteins, their resistance to denaturing influences, organic solvents, and change ligand-binding properties. Protein engineering allows, by replacing amino acids, to improve the functioning of enzymes and their specificity, change the optimal pH values at which the enzyme operates, eliminate unwanted side activities, eliminate sections of molecules that inhibit enzymatic reactions, increase the effectiveness of protein drugs, and so on. For example, replacing only one threonine residue with an alanine or proline residue made it possible to increase the activity of the enzyme tyrosyltRNA synthetase by 50 times, and thanks to the replacement of 8 amino acid residues, the so-called thermolysin-like protease from Bacillus stearothermophilus acquired the ability to remain active at 120 ° C for several hours . Protein engineering also includes work on targeted changes in the properties of proteins using chemical modifications, for example, the introduction of photoactivated compounds that change the properties of the molecule under the influence of light, tag compounds that allow tracking the paths of protein movement in the cell or directing it to various components of the cell, etc. similar. Such work is carried out mainly on recombinant proteins obtained using genetic engineering methods.

Protein engineering can be divided into two areas: rational design and directed molecular evolution of proteins. The first involves the use of information about the structure-function relationships in proteins, obtained using physicochemical and biological methods, as well as computer molecular modeling, in order to determine which changes in the primary structure should lead to the desired result. Thus, to increase the thermal stability of a protein, it is necessary to determine its spatial structure, identify “weak” areas (for example, amino acids that are not strongly connected to their environment), and select the best options for replacing them with other amino acids using molecular modeling and optimizing the energy parameters of the molecule; after this, mutate the corresponding gene, and then obtain and study the mutant protein. If this protein does not meet the specified parameters, a new analysis is performed and the described cycle is repeated. This approach is most often used in the case of constructing artificial proteins (de novo proteins) with specified properties, when the input is a new amino acid sequence, mainly or completely specified by a person, and the output is a protein molecule with the desired characteristics. So far, however, in this way it is possible to obtain only small proteins de novo with a simple spatial structure and introduce simple functional activities into them, for example, metal-binding sites or short peptide fragments that carry some biological functions.

In the directed molecular evolution of proteins using genetic engineering methods, a large set of different mutant genes of the target protein is obtained, which are then expressed in a special way, in particular on the surface of phages (“phage display”) or in bacterial cells, in order to make selection of mutants possible with the best characteristics. For this purpose, for example, the genes of the desired protein or its parts are inserted into the phage genome - into the gene encoding the protein located on the surface of the phage particle. Moreover, each individual phage carries its own mutant protein, which has certain properties for which selection is made. Mutant genes are produced by “mixing” a set of genes for similar natural proteins from different organisms, usually using the polymerase chain reaction method, so that each resulting mutant protein may include fragments of many “parent” proteins. Essentially, this approach mimics the natural evolution of proteins, but at a much faster pace. The main task of a protein engineer in this case is to develop an effective selection system that will allow selecting the best mutant versions of proteins with the required parameters. In the case of the above-mentioned task - to increase the thermal stability of a protein - selection can be carried out, for example, by growing cells containing mutant genes at elevated temperatures (provided that the presence of a mutant protein in the cell increases its thermal stability).

Both of these areas of protein engineering have the same goal and complement each other. Thus, the study of mutant protein variants obtained using molecular evolution methods allows us to better understand the structural and functional organization of protein molecules and use the acquired knowledge for the targeted rational design of new proteins. The further development of protein engineering makes it possible to solve many practical problems in improving natural proteins and obtaining new ones for the needs of medicine, agriculture, and biotechnology. In the future, it is possible to create proteins with functions unknown in living nature.

Lit.: Brannigan J.A., Wilkinson A.J. Protein engineering 20 years on // Nature Reviews. Molecular Cell Biology. 2002. Vol. 3. No. 12; Patrushev L.I. Artificial genetic systems. M., 2004. T. 1: Gene and protein engineering.

Genetic engineering of proteins, hybrid proteins, viral vectors. Reporter proteins in fusion proteins

Peptide and Epitope Libraries

The role of TNF in the pathogenesis of rheumatoid arthritis and other autoimmune diseases

Study of the interaction between Vhh41 and human TNF using surface plasma resonance

Study of the biological properties of the fluorescent sensor Vhh41-KTNFin vitro and in vivo

Production and characterization of a new recombinant single domain antibody that specifically binds to human TNF but does not block its biological activity