New genetically engineered proteins based on recombinant anti-TNF antibodies Efimov Grigory Aleksandrovich. Methods of genetic engineering in obtaining recombinant proteins Genetically engineered proteins
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's genetic engineering is a system of experimental methods that make 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 manipulating nucleic acid molecules outside the cell and transferring the created gene constructs to a living organism, which results in their inclusion and activity in this organism and in its offspring. We are talking about directed, according to a predetermined program, the 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 new unique genetic, biochemical, and then physiological properties to it.
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 that are useful for humans. For example, obtaining "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 carry out 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 of a purified DNA fragment, which allows you 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 by 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 with the help of a number of enzymes, primarily restriction enzymes. Over 400 different restrictases are currently in use. These enzymes synthesize a wide variety of microorganisms.
Restriction enzymes recognize and cleave specific nucleotide sequences in the double-stranded DNA molecule. However, restriction enzymes alone are not enough for molecular cloning, because the hydrogen bonds between the four bases that form the sticky ends are not strong enough to hold the two combined DNA fragments.
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 recombinant DNA to replicate in the cell.
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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 design.
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, a more detailed knowledge of the structure and function of proteins, as well as advances in high technology, may greatly expand the possibilities of protein engineering. As a result, even non-natural amino acids can be included thanks to a new method that allows new amino acids to be included in 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 desired characteristics. A natural way to implement such a task is to predict the structure of the gene encoding the modified protein, the implementation of its synthesis, cloning and expression in recipient cells.
The first controlled modification of a protein was carried out in the mid-1960s by Koshland and Bender. To replace the hydroxyl group with a sulfhydryl group in the active center of the protease, subtilisin, they used the method of chemical modification. However, as it turned out, such thiolsubtilisin does not retain protease activity.
Protein in chemical terms is a molecule of the same type, 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 themselves the question: is it possible to design completely new amino acid sequences so that they perform the functions that a person needs much better than ordinary proteins? The name Protein Engineering came up for this idea.
They began to think about such engineering back in the 50s of the XX century. This happened immediately after the decoding of the first protein amino acid sequences. In many laboratories of the world, attempts have been made to duplicate nature and chemically synthesize polyamino acid sequences given absolutely arbitrarily.
Most of all, the chemist B. Merrifield succeeded in this. This American managed to develop an extremely efficient method for the synthesis of polyamino acid chains. Merrifield was awarded the Nobel Prize in Chemistry in 1984 for this.
Figure 1. Scheme of the functioning of protein engineering
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 exactly reproduce the amino acid sequences, that is, it was wrong. 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 exactly the same.
There was also another problem. Even those molecules that the robot synthesized correctly did not take the spatial form that is necessary for the functioning of the enzyme. Thus, the attempt to replace nature with the usual methods of organic chemistry has led to very modest success.
Scientists had to learn from nature, looking for the necessary modifications of proteins. The point here is that mutations are constantly occurring in nature, leading to a change in the amino acid sequences of proteins. If we select mutants with the necessary properties that process this or that substrate more efficiently, then it is possible to isolate from such a mutant an altered enzyme, due to which the cell acquires new properties. But this process takes a very long time.
Everything changed when genetic engineering appeared. Thanks to her, they began to create artificial genes with any sequence of nucleotides. These genes were inserted into prepared vector molecules and these DNAs were introduced into bacteria or yeast. There, a copy of the RNA was removed from the artificial gene. As a result, the desired protein was produced. Errors in its synthesis were excluded. The main thing was to choose the right DNA sequence, and then the enzymatic system of the cell itself did its job flawlessly. Thus, we can conclude that genetic engineering has opened the way for protein engineering in its most radical form.
Protein engineering strategies
Targeted protein modification. In targeted modification of a protein, the scientist uses detailed knowledge of the protein's structure and function to make the desired changes. Generally, this method has the advantage of being inexpensive and technically uncomplicated, since site-directed mutagenesis techniques are well developed. However, its main disadvantage is that information about the detailed structure of a protein is often missing, and even when the structure is known, it can be very difficult to predict the impact of different mutations.
Protein modification software algorithms seek to identify new amino acid sequences that require little energy to form a predetermined target structure. While the sequence to be found is large, the most challenging requirement for protein modification is a fast, yet precise, way to identify and determine 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. Further rounds of mutation and selection are applied. This method mimics natural evolution and generally gives excellent results for directed modification.
An additional technique, known as DNA shuffling, mixes and brings out parts of successful variants for 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 needed, to be able to predict what impact a given mutation will have. Indeed, the results of directed evolution experiments are surprising, since 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. A large amount of recombinant DNA must be mutated and the products must be screened for the desired quality. The sheer number of options often require the purchase of robotics to automate the process. In addition, it is not always easy to screen for all traits of interest.
In the peptide libraries discussed above, the latter are covalently linked to the carrier protein. In this form, they are one of the representatives of hybrid proteins obtained by genetic engineering.
In another case, fusion proteins are used to obtain a high level of expression of short peptides in bacterial cells due to the stabilization of these peptides within the fusion proteins. Fusion proteins are often used to identify and purify difficult-to-detect recombinant proteins. For example, by attaching -galactosidase as a reporter protein to the C-terminus of the protein under study, it is possible to purify the recombinant protein by the activity of -galactosidase, determining its antigenic determinants by immunochemical methods. By linking DNA fragments containing open reading frames (ORFs) with reporter protein genes, it is possible to purify such fusion proteins for reporter protein activity and use them for immunization of 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 cloned ORF to be linked in the same reading frame as the reporter gene. The hybrid proteins resulting from the expression of these recombinant genes are identified using antibodies by enzyme immunoassay methods. Hybrid genes that combine secreted proteins and reporter proteins make it possible to explore the mechanisms of secretion in a new way, as well as the localization and movement of secreted proteins in tissues.
Hybrid toxins
A series of works by I. Pastan and his collaborators on the design of targeted hybrid toxins perfectly illustrates the possibilities of protein engineering in terms of combining different functional domains of proteins to achieve specific biological effects.
Rice. II.22. Targeted drugs based on hybrid toxins
a- a generalized scheme of the structure of a drug with directed action; b– the structure of the pseudomonadic toxin (numbers indicate the position of amino acid residues); in– the structure of the hybrid toxin; G– hybrid toxin based on monoclonal antibodies
An ideal drug with a strictly specific selective action should have at least the following structural and functional features (Fig. II.22, a). Such a drug must contain an active principle to achieve a physiological effect and a ligand that recognizes a receptor on the surface of target cells. In addition, it must contain structural elements recognized by the body's transport system for drug delivery to target cells, as well as a spacer site necessary to separate the active principle from the remaining functional parts of the drug after it has been delivered to the address. It is this ideal scheme that is realized in the natural exotoxin of Pseudomonas aeruginosa. P. aeruginosa exotoxin A is a protein consisting of a single polypeptide chain 613 amino acids long, which is organized into three functional domains (see Fig. II.22, b). The N-terminal domain Ia (amino acid residues 1–252) is required for interaction with the surface of target cells (a prototype ligand for an ideal targeted drug). The functions of domain Ib (amino acid residues 365–404) are currently unknown. Domain II (amino acid residues 253-364) ensures efficient transfer of the toxin to the cell cytosol (drug transport system), and domain III (amino acid residues 405-613) carries out ADP-ribosylation of the translation elongation factor EF2, which leads to suppression of translation and cell death- targets. Thus, for exotoxin A to have a cytotoxic effect, it is necessary to recognize receptors on the cell surface using domain Ia, penetrate into the cell using receptor-mediated endocytosis, and be translocated through the inner membrane into the cytosol, where the EF2 factor is localized. The main idea in creating targeted toxins was to replace domain Ia with some other peptide ligand that interacts with another group of receptors on the cell surface, and thereby change the specificity of the action of the toxin in relation to the cells themselves (see Fig. II. 22, in).
It was found that the removal of domain Ia by genetic engineering methods dramatically (by hundreds and thousands of times) reduces the toxicity of such a truncated protein both in relation to cells of various lines and in vivo. Attachment to the C-terminal part of the truncated polypeptide of the human interleukin 2 molecule was carried out by combining the structural parts of the corresponding genes in the expression vector. The purified hybrid toxin turned out to be extremely toxic to cells carrying interleukin 2 receptors on their surface, and did not act on cells that lacked these receptors and that died under the action of natural toxin. Internalization(translocation into cells) of the hybrid toxin was mediated by the p55 and p70 subunits of the interleukin 2 receptor. Thus, as a result of the action of the hybrid toxin on a population of cells, some of which express interleukin 2 receptors on their surface, selective death of these cells occurs.
In the body, most resting and memory T cells do not express high-affinity interleukin 2 receptors on their surface, while T cells stimulated with alloantigens contain such receptors. Therefore, intraperitoneal administration of the hybrid toxin to rats with experimental arthritis, a disease caused by pathological T-cell activation, reduced the symptoms of the disease. The hybrid toxin significantly reduced transplant rejection in mice as well.
These pioneering works were followed by a whole series of studies aimed at creating similar systems for targeted delivery of various cytotoxic polypeptides. In the process of further improvement of the system of targeted delivery of the pseudomonas toxin using interleukin 2 as a ligand, they abandoned the complete removal of the target domain of the toxin and limited it to its inactivation by introducing four site-specific mutations into the toxin gene. Molecules of this hybrid toxin turned out to be 10–100 times more effective cytotoxic agents against human and monkey cells expressing receptors for interleukin 2 on their surface, and also had a significantly longer half-life in the blood of mice in vivo compared to the previously obtained construct.
On the basis of Pseudomonas toxin, hybrid toxins were created containing polypeptide chains of interleukin 4, interleukin 6, transforming growth factor type and insulin-like growth factor I as ligands. Highly specific cytotoxicity against tumor cells (including human myeloma cells) was shown for all these hybrid proteins. ) with corresponding receptors. The use of a part of the CD4 polypeptide chain as a ligand in the hybrid toxin, a glycoprotein of the surface of T cells, which is the receptor of the HIV virus and interacts with its glycoprotein gp120, made it possible to selectively infect T cells infected with the HIV virus and expressing the viral gp120 protein on their surface.
The same principle of suppression of infection caused by HIV viruses by soluble CD4 receptors was used in the construction of hybrid proteins that combine parts of CD4 polypeptide chains with constant parts of heavy or light chains of human immunoglobulins. At the same time, in the process of combining genes, the nucleotide sequences encoding the transmembrane and cytoplasmic domains of CD4, as well as the variable part of the polypeptide chains of immunoglobulins, were removed. The resulting hybrid molecules, called immunoadhesins, due to the constant part of the immunoglobulin molecule, they acquired increased stability in the body and, in addition, retained specific properties mediated by the constant parts of immunoglobulins: binding of the Fc receptor and protein A, the ability to fix complement and transfer through the placental barrier. The combination of all these properties made it possible for immunoadhesins to effectively interrupt the infection of T-cells with the HIV-I virus, blocking both the virus itself and the cells infected by it, expressing the gp120 viral antigen on their surface.
Further improvement of genetically engineered constructs based on Pseudomonas exotoxin A occurred after the variable domains of monoclonal antibodies to the p55 component of the human interleukin 2 receptor began to be used as the target part of the hybrid toxin. In this recombinant protein, a 15-mer peptide amino acid linker was used to connect the variable domain of the heavy chain of this immunoglobulin to the variable domain of its light chain, and the C-terminus of the light chain to the N-terminus of the truncated Pseudomonas toxin (see Fig. II.22, G). Such hybrid toxin molecules also turned out to be highly specific cytotoxic agents against human leukemic cells expressing interleukin 2 receptors on their surface.
The developed approach demonstrated the possibility of using specific antibodies as target parts of hybrid toxins. This provides researchers with a universal method of targeted delivery of toxins, which in the future will make it possible to exert a cytotoxic effect on any groups of cells expressing specific antigens on their surface, i.e. significantly expand the number of targets for chemotherapeutic effects using recombinant proteins.
In addition to Pseudomonas exotoxin A, diphtheria toxin, tumor necrosis factor, and the ricin A chain have been successfully used as the active principle in hybrid toxins. Since the A-protein interacts selectively with the constant (Fc) portions of the G-class immunoglobulins of many mammals, such a hybrid toxin, paired with an immunoglobulin prepared against an antigen on the cell surface, selectively binds to and kills these cells. Such immunotoxins are another potential antitumor agent and can be used against cells expressing specific antigens on their surface.
Rice. II.23. Use of a fusion protein to regulate gene expression
Genetic engineering methods open up endless possibilities for constructing new proteins by combining various functional domains of polypeptide chains in various combinations. The production of targeted hybrid toxins illustrates the possibilities of such an approach in protein engineering. As a final illustration of the possibilities of this group of methods, let us consider a hybrid protein as a new regulator of gene activity. When constructing such a protein by genetic engineering, the DNA-binding domain in the glucocorticoid hormone receptor was replaced with the corresponding domain of the E. coli LexA repressor (Fig. II.23).
Introduction of the gene operator sequence lexA into the promoter region of the globin gene (or other genes) led to the activation of the promoter under the action of a hybrid protein in the presence of dexamethasone, a synthetic hormone that interacts with the glucocorticoid receptor. Thus, in the new genetic environment, the nucleotide sequence of the gene operator lexA E. coli functioned as a transcriptional enhancer in the presence of a fusion activator protein recognizing this sequence. The results of the work demonstrate the possibility of creating new proteins - regulators of gene activity by combining known functional domains.
The development of protein engineering is largely constrained by a lack of knowledge about the structural and functional relationships in proteins, which is due to the complexity of the object of study. Numerous works aimed at the study of such relationships, as a rule, are empirical in nature and end with the localization of amino acids essential for the functioning of the active sites of enzymes. Therefore, the main task of protein engineering - to obtain a protein with desired properties from a known sequence of amino acid residues - is currently still far from being resolved. Nevertheless, even now it is sometimes possible to purposefully change some properties of existing enzymes by replacing a small number of amino acid residues of their polypeptide chains using site-directed mutagenesis.
480 rub. | 150 UAH | $7.5 ", MOUSEOFF, FGCOLOR, "#FFFFCC",BGCOLOR, "#393939");" onMouseOut="return nd();"> Thesis - 480 rubles, shipping 10 minutes 24 hours a day, seven days a week and holidays
Efimov Grigory Alexandrovich. New genetically engineered proteins based on recombinant antibodies against TNF: dissertation... Candidate of Biological Sciences: 03.01.03 / Efimov Grigory Aleksandrovich; [Place of defense: Institute of Molecular Biology named after V.A. - 122 p.
Introduction
Literature review 9
1. History of opening tnf 9
2. Superfamily tnf 10
3. System structure tnfnfr 12
4. tnf functions 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 anti-inf therapy 23
8. New approaches and perspectives for tnf blocking 25
Materials and methods of research 29
1. Preparation and characterization of a novel camel single-domain anti-human tnf antibody 29
Expression and purification of single domain antibody Vhh41 29
Evaluation of the binding of the Vhh41 antibody to human TNF by ELISA 30
Study of the interaction of Vhh41 and human TNF by surface plasma resonance 31
Studies on the ability of Vhh41 to block human TNF 31
2. Construction, production and characterization of hybrid proteins of TNF fluorescent sensors 32
Construction of genes encoding TNF sensors. 32
Expression and purification of fluorescent TNF sensors. 33
Analysis of the interaction of Vhh41-K with recombinant TNF. 34
Investigation of the biological properties of the fluorescent sensor Vhh41-KTNFin vitro and in vivo. Z5
Study of the ability of a fluorescent sensor to bind TNF in vivo 36
In vivo study of TNF expression using the resulting fluorescent sensor...39
3. Production and characterization of a single-chain ANTINF antibody 40
Study of mouse monoclonal antibody F10 40
Construction and expression of single chain antibody ahT-4 41
Measuring the biological activity of the ahT-4 single chain antibody 42
4. Preparation and characterization of chimeric anti-inf antibody 43
5. Construction, preparation and characterization of A9 and MA9 bispecific antibodies 43
Construction, expression and purification of antibodies A9 and tA9 43 Interaction of antibodies A9 and tA9 with recombinant human TNF method
surface plasma resonance 44
Cytotoxic test 45
Cytofluorimetry 45
Evaluation of the ability of the bispecific antibody A9 to retain human TNF for
surface of macrophages 45
6. Comparative evaluation 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. Obtaining 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. Construction, production and characterization of TNF molecular sensors for in vivo study of tnf expression based on single domain recombinant antibodies and red fluorescent protein 56
Obtaining genetic constructs encoding the TNF Vhh41-Ku fluorescent sensor
control fusion proteins 56
Expression and purification of the TNF Vhh41-K fluorescent sensor. 57
Analysis of the interaction of the fluorescent sensor TNF Vhh41-K with recombinant mouse TNF
and human 58
Study of the biological properties of the fluorescent sensor TNF Vhh41-Kin vitro and in vivo. 61
Study of the ability of a fluorescent sensor to bind TNF in vivo 66
In vivo study of TNF expression using the obtained fluorescent sensor... 69
3. Preparation and characterization of a recombinant single chain antibody blocking 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 the lung and
heavy chains of mouse monoclonal antibody F 10 74
Measurement of the activity of single-chain antibody ahT-4 75
4. Development and analysis of a chimeric anti-human TNF antibody
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 that of
infliximab in vitro 79
Analysis of the activity of the chimeric antibody 13239 in vivo 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 A9 and mA9 antibodies with recombinant human TNF 86
Antibodies A9 and rA9 block TNF-dependent cytotoxicity in vitro 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 series in vivo 96
Comparative evaluation of the effectiveness of targeted blocking of TNF produced by cells of the monocyte-macrophage series 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 of anticytokine therapy was carried out in 1985, when mice were injected with polyclonal antiNF rabbit serum, which prevented the development of lethal hepatotoxicity induced by LPS administration. Similar results were obtained in monkeys: baboons injected with a mouse monoclonal antibody against human TNF survived after an intravenous injection of a lethal dose of E. coli [104].
The first therapeutic TNF blocker was developed from a high affinity mouse monoclonal antibody A2 derived from mice immunized with human TNFα. Because antibodies of other species have significant differences in amino acid sequence, they are unsuitable for long-term therapeutic use in humans. Therefore, by 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 anti-TNF antibody received an international non-proprietary name - infliximab.
One of the most obvious applications of antiNF therapy has been in the treatment of sepsis. However, clinical studies have not shown significant results, which, apparently, is due to the fact that by the time the clinical picture of sepsis develops, irreversible signaling cascades are already running.
By this time, a lot of evidence had already been accumulated indicating the involvement of TNF in the pathogenesis of rheumatoid arthritis, so this disease was chosen as the next potential target for antiNF therapy. Pilot studies with infliximab in rheumatoid arthritis have shown promising results, and further randomized, double-blind trials have confirmed the efficacy of antiNF therapy in the treatment of autoimmune diseases. However, after repeated injections, some patients developed antibodies specific for 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 doses of methotrexate, a cytotoxic drug used for monotherapy in RA. In combination, 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. Against the background of the introduction of TNF inhibitors, the balance of T-effector and T-regulatory cells shifts.
AntiNF therapy is not an etiotropic therapy and theoretically should be used throughout the patient's life, however, in some cases, it is possible to achieve a stable remission, which persists even after antiNF therapy is discontinued.
TNF blockers have shown their effectiveness in the treatment of other autoimmune and inflammatory diseases: it has been shown that TNF plays a significant role in the pathogenesis of Crohn's disease - it is overexpressed in inflamed areas of the intestine. Preliminary successes in the treatment of refractory Crohn's disease with infliximab were later confirmed in randomized clinical trials, resulting in the approval of infliximab for this disease as well.
The pathogenesis of ankylosing spondylitis (Bekhterev's disease), another chronic systemic autoimmune disease predominantly affecting the joints, is also due to TNF overexpression. Clinical trials of infliximab have been successful for this disease as well. In addition, antiNF therapy has shown high efficacy in the treatment of psoriasis and psoriatic arthritis.
To date, infliximab and other TNF blockers have been 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 supported by laboratory animal experiments. The introduction of TNF aggravated the symptoms of experimental autoimmune encephalomyelitis in rats, and the introduction of antiNF antibodies prevented the development of this disease.
However, clinical trials in 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 whose pathogenesis is similar to that 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, an increase in the number of foci in magnetic resonance imaging.
The success of infliximab has given impetus to the development of new molecules capable of blocking signal transduction through the 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 action of infliximab and made patients refractory to therapy. To overcome this limitation, a route was chosen 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 constructed from soluble TNFR2. Its development was based on data that a soluble form of the second TNF receptor is present in the human body. Desquamated by metalloproteases, TNFR2 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 fragment of IgG1 immunoglobulin. Binding to the constant region of an antibody significantly increases the systemic half-life of the drug by recycling the protein through the FcRn receptor. The neutralizing activity of the fusion protein was demonstrated in both in vitro and in vivo experiments, and later confirmed in clinical trials in patients with rheumatoid arthritis.
However, in the treatment of inflammatory bowel disease, etanercept, unlike infliximab, has not shown therapeutic efficacy. An experimental TNF blocker, onercept, based on another TNF receptor, TNFR1 (p55), despite encouraging pilot clinical studies in a randomized, placebo-controlled, double-blind study, also did not show efficacy 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 block TNF, only infliximab binds to T cells in the lesion and induces apoptosis in them. This may explain the difference in the effectiveness of antibody-based and recombinant receptor-based blockers in inflammatory bowel disease.
Study of the Interaction of Vhh41 and Human TNF by Surface Plasma Resonance
The genetic construct encoding the A9 bispecific antibody was assembled by a 4-primer GAP reaction similar to that described above for the ahT-4 single chain antibody gene. The resulting sequence consisted of: the single domain antiNF antibody gene, then the sequence encoding the (Gly4Ser)3 species linker, and the single chain anti-P4/80 antibody gene (courtesy of S. Gordon and M. Stacey). Restriction recognition sites Ncol and Xhol were included in the sequence of forward and reverse primers, respectively. After restriction PCR product and cloning it into the expression vector pET-28b (Novagen), the sequence encoding the polyhexidine tag was at the 3 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 shA9 were used to transform E. coli cells of the Rosetta2(DE3)pLysS strain (Novagen). The best producer 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 the logarithmic phase, and then expression was induced by 0.2 mM IPTG. After 4 hours, the cultures were subjected to centrifugation at 3200 g for 30 min. The pellets were frozen and then resuspended in lysis buffer (50 mM TrisHCl, 300 MMNaCl, 5% glycerol, 0.5% Triton X-100 detergent, 10000 U/mL lysozyme, 10 mM P-mercaptoethanol) and then disintegrated with 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 clarified supernatants on a chromatographic column containing agarose conjugated with 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 the 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 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, followed by Coomassie staining.
Interaction of A9 and mA9 antibodies with recombinant human TNF by surface plasmon resonance.
Comparison of the affinities and kinetics of the interaction of A9 and mA9 antibodies with recombinant human TNF was carried out on a ProteOn XPR36 instrument (Bio-Rad). In the course of measuring all interactions, phosphate-buffered saline (pH = 7.4) was used, to which Tween 20 detergent was added to a concentration of 0.005%, the surface temperature of the chip 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 doubly decreasing concentrations (50 -3 nM) was applied to five parallel channels. The buffer containing no antibodies was introduced into the sixth channel for normalization. The analysis of the received sensograms was carried out in the ProteOn Manager (Bio-Rad) program using the Langmuir model.
In experiments with peritoneal macrophages, peritoneal cavity cells were isolated from wild-type (C57BL/6) mice and immediately stained using fluorochrome-cojugated antibodies. To obtain bone marrow macrophages, the bone marrow was isolated, after which the cells were cultured for 10 days in a 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 with fluorochrome. 2) monoclonal mouse antibodies to the hexahistidine sequence (Novagen - 70796), then with secondary antibodies to mouse IgG conjugated with fluorochrome. 3) recombinant human TNF was added to the cells, followed by monoclonal antiNF antibodies (Miltenyi Biotec - clone: cA2) labeled with fluorochrome.
In addition, cells were stained with anti-P4/80 and anti-CD 1 lb antibodies conjugated to fluorochromes. Samples were analyzed either on a F ACS Aria instrument (BDBiosciences) or a Guava EasyCyte 8HT (Millipore) and then processed using the FlowJo software (Treestar Inc.).
Evaluation of the ability of the A9 bispecific antibody to retain human TNF on the surface of macrophages.
Peritoneal macrophages from mice producing human TNF were isolated and seeded at 100,000 cells per well in 96-well culture plates. The cells were incubated for 2 h at 37C, 5% CO2, after which the non-attached cells were washed off with warm phosphate buffer. The cells were then incubated overnight at 37°C, 5% CO2. After washing with 200 µl of warm DMEM medium, the cells were incubated with A9 antibodies at a concentration of 2 µg/ml or with DMEM medium for 30 minutes at 37°C. After another wash, the cells were stimulated with LPS (Sigma, L2630) at a concentration of 100 ng/ml. After 4 hours, 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 mice producing human TNF was isolated, after which the cells were cultured for 10 days in a conditioned medium (obtained on line L929), then the cells were removed from the plastic with ice-cold phosphate buffer. The number of live cells was counted and they were seated on 96 well plates at a concentration of 50,000 cells/well. Then 250 uM A9 antibody or hTNF-VffH single domain antibody or blank medium (DMEM) was added to the cells. Cells were incubated with antibodies for 30 min. The wells were then washed with phosphate-buffered saline. After that, TNF production was stimulated by LPS (Sigma - L2630) at a concentration of 100 ng/ml. After 4 hours, the supernatants were collected, the concentration of TNF in them was measured using a cytotoxic test on the line of mouse fibrosarcoma L929 according to the 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 on mouse lines in which the Tnf gene is deleted in separate cell populations, a hypothesis was formulated about 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 by myeloid cells, has a unique protective function. In addition, data have been obtained in our laboratory indicating the pathogenic properties of TNF from myeloid cells in autoimmune diseases. The therapeutically applied complete blocking of PMB does not take into account these features. As part of the development of this hypothesis, specific inhibition of TNF produced by cells of the monocyte-macrophage series was chosen, which could have a significant advantage over the 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 antiNF therapy effective in those diseases for which TNF blockers have not previously shown clinical efficacy, or even caused an increase in symptoms. In addition, this approach could potentially reduce the required dose through targeted delivery to producer cells.
To test this assumption, we designed and tested a bispecific antibody that binds with one part of the macrophage surface due to interaction with the F4/80 transmembrane molecule, and with the other specificity captures and blocks the TNF produced by them.
Molecular cloning, expression and purification of bispecific antibodies. The bispecific antibody, a selective blocker of macrophage TNF, was named A9. A single-domain blocking antiNF antibody hTNF-VffH and a single-chain antibody (scFv) against macrophage surface marker F4/80 (kindly provided by S. Gordon (Oxford University, UK) and M. Stacey (University of Leeds, UK) were used to create a genetic construct encoding it. The sequences encoding both antibodies were amplified by polymerase chain reaction (PCR) and cloned into an expression vector so that they were in the same reading frame, and a nucleotide sequence encoding a flexible glycine-serine linker (GSGGGGSG) was formed between them. The sequence encoding the histidine hexamer is located at the C-terminus of the sequence for subsequent purification of the protein (Fig. 31).
The design of the bispecific A9 antibody, a schematic representation of its mechanism of action, the structure of genetic constructs encoding the bispecific A9 antibody and the control systemic blocker of TNF, the tA9 antibody. (A) The A9 bispecific antibody consists of a single domain antibody (VHH) against human TNF and a single chain antibody (scFv) against the F4/80 surface molecule expressed on monocytes and macrophages. (B) The 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) Diagram of the genetic construct of the A9 bispecific antibody and the control TNF systemic blocker, tA9. The single-domain anti-NF 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 a histidine hexamer for affinity purification. The control tA9 antibody has a similar sequence, except that the 6 hypervariable regions of the anti-P4/80 antibody are replaced with sequences of the (Gly3Ser)n type, 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 the effects associated with differences in antibody affinity, it was decided to use a blocker having 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 under study. 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 anti-P4/80 scFv are replaced by sequences of the type (Gly3Ser)n, the same length as original CDR regions (see Fig. 31B).
Both antibodies were expressed in the bacterial system and purified by affinity chromatography.
The size of the A9 antibody, determined by electrophoretic mobility and HPLC data, corresponded to the calculated molecular weight of 45 kDa (Fig. 32). chromatography. On the left - the values of the molecular weight of proteins. (B) Chromatogram of the A9 bispecific antibody (marked in red) superimposed on the chromatogram of molecular weight markers. (B) Function of transit time of a molecule as a function of molecular weight. The calculated molecular weight of the A9 bispecific antibody was 43.4 kDa.
Interaction of A9 and tA9 antibodies with recombinant human TNF. The interaction kinetics of A9 and mA9 antibodies 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 was measured on a ProteOn XPR36 instrument. 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 the A9 antibody 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 interaction kinetics of A9 and mA9 antibodies 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 was measured on a ProteOn XPR36 instrument. 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 the A9 antibody to retain bound TNF.
Kinetics of the interaction of the bispecific antibody A9 and the 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 A9 bispecific antibody and the control tA9 antibody were immobilized are shown. The abscissa shows the time in seconds, the ordinate shows the resonance angle shift in conventional units (AU). (B) For each group of sensograms, the binding rate (op-rate), dissociation rate (off-rate), and dissociation constant (Kd) were calculated. The resulting mean values as well as the standard deviation (SD) are plotted on an isoaffinity diagram. The diagonal lines correspond to the indicated values of the dissociation constant.
To evaluate the relative activity of the A9 antibody in inhibiting the biological effects of TNF, a cytotoxic text was performed on the L929 mouse fibrosarcoma line. Serial dilutions of A9 and mA9 antibodies were added to constant concentrations of recombinant human TNF and actinomycin-D. According to the obtained data, antibodies A9 and rA9 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 anti-NF antibody hTNF-VffH, which is part of A9 and mA9 (Fig. 34 B). 10 10 10
AntiNF activity of the A9 bispecific antibody, mA9 control antibody, and hTNF-VffH single domain antibody. (A) Comparison of the activity of the A9 bispecific antibody and the control tA9 antibody. The survival curve of L929 mouse fibrosarcoma cells under simultaneous exposure to a constant dose of human TNF and decreasing doses of antibodies A9 and tA9 is shown. (B) Comparison of the activity of the A9 bispecific antibody and the hTNF-VnH single domain antibody. The survival curve of L929 mouse fibrosarcoma cells is shown under simultaneous exposure to a constant dose of human TNF and decreasing doses of A9 and hTNF-VHH antibodies. 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 mA9 to the surface of macrophages through interaction with the surface molecule F4/80.
The ability of the A9 bispecific antibody 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, and at the same time specific staining for the bispecific A9 antibody was carried out through antibodies to VHH OR antibodies to the polyhistidine label. The samples were then subjected to flow cytometry and analysis.
These experiments showed that the A9 bispecific antibody was 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). The decrease in the level of parallel anti-F4/80 staining with the addition of the A9 antibody due to the competition of two antibodies for binding to the target confirms that A9 specifically interacts with this particular molecule on the cell surface (Fig. 35 G and 3).
The name Mac-1 is also used. A constituent element of the C3 receptor component of the complement system. In mice, it is expressed on monocytes, macrophages, and microglial cells. bispecific antibody
Cells of the peritoneal cavity were incubated with or without the A9 bispecific antibody (shown in red) or without it (shown in blue), and then stained with fluorescently labeled antibodies to surface markers specific for monocyte-macrophage cells, as well as antibodies specific to A9. Then the obtained samples were analyzed by flow cytometry. (A, C, E, G) staining through antibodies to the VHH domain. (B, D, F, 3) staining through antibodies to the polyhexidine sequence. (A, B) A9 bispecific antibody binds to cells selected for high F4/80 and CD1 lb expression (macrophages). On the displayed histogram, the horizontal axis shows the fluorescence value in the staining channel at A9, 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 at A9, the vertical axis shows the fluorescence value in the staining channel at F4/80. (E, E) -bispecific antibody A9 does not bind to cells of the peritoneal cavity that do not express F4/80 and CD1 lb (lymphocytes). On the displayed histogram, the horizontal axis shows the fluorescence value in the staining channel at A9, the vertical axis shows the normalized frequency of occurrence of the event. (G, 3) -incubation with the A9 bispecific antibody reduces the staining intensity by F4/80. On the depicted histogram, the horizontal axis shows the fluorescence value in the staining channel at F4/80, the vertical axis shows the normalized frequency of occurrence of the event.
Bone marrow macrophages were incubated with A9 bispecific antibody (shown in red), without it (shown in blue), or with control tA9 antibody (shown in black) and then stained with antibodies specific for A9/mA9. The obtained samples were analyzed by flow cytometry. (A) A9 bispecific antibody binds specifically to bone marrow macrophages. On the displayed histogram, the horizontal axis shows the fluorescence value in the staining channel at A9, the vertical axis shows the normalized frequency of occurrence of the event. (B) The mA9 control antibody is unable to bind to bone marrow macrophages. On the displayed histogram, the horizontal axis shows the value of fluorescence in the staining channel on wA9, the vertical axis shows the normalized frequency of occurrence of the event.
In addition, in additional cytofluorometric experiments, it was shown 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 binding of the two antigens simultaneously is sterically possible.
Cells of the peritoneal cavity were incubated with or without A9 bispecific antibody (shown in red) or without it (shown in blue), then with recombinant human TNF, after which they were 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) A9 bispecific antibody capable of retaining human TNF on the surface of macrophages (cells selected for high expression of F4/80 and CD1 lb). On the displayed histogram, the horizontal axis shows the fluorescence value in the TNF staining channel, 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, the vertical axis shows the fluorescence values in the F4/80 staining channel.
PROTEIN ENGINEERING, a branch of molecular biology and bioengineering, whose tasks include the purposeful change in the structure of natural proteins and the production of new proteins with desired 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 to change the structure of genes in a certain way 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 directed changes in their structure. With the help of protein engineering, it is possible to increase the thermal stability of proteins, their resistance to denaturing effects, organic solvents, and to change their 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 works, eliminate unwanted side activities, eliminate molecular regions that inhibit enzymatic reactions, increase the effectiveness of protein drugs, and so on. For example, the replacement of only one threonine residue with an alanine or proline residue allowed a 50-fold increase in the activity of the tyrosyl-tRNA synthetase enzyme, and due to the replacement of 8 amino acid residues, the so-called thermolysin-like protease from Bacillus stearothermophilus acquired the ability to maintain activity at 120 °C for several hours. . Protein engineering also includes works on directed changes in the properties of proteins using chemical modifications, for example, the introduction of photoactivated compounds that change the properties of a molecule under the action of light, label compounds that make it possible to trace the pathways of a protein in a cell or direct it to various components of a cell, and similar. Such work is carried out mainly on recombinant proteins obtained using genetic engineering methods.
Two directions can be distinguished in protein engineering: rational design and directed molecular evolution of proteins. The first involves the use of information about the structural-functional 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. So, 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 with their environment), select the best options for substitutions for other amino acids using molecular modeling and optimization of the energy parameters of the molecule; after that, mutate the corresponding gene, and then obtain and analyze the mutant protein. If this protein does not meet the specified parameters, conduct a new analysis and repeat the described cycle. This approach is most often used in the case of constructing artificial proteins (de novo proteins) with desired properties, when the input is a new amino acid sequence, mostly or completely specified by a human, and the output is a protein molecule with the desired characteristics. So far, however, only small de novo proteins with a simple spatial structure can be obtained in this way and simple functional activities can be introduced into them, for example, metal-binding sites or short peptide fragments that carry any biological functions.
In the directed molecular evolution of proteins, a large set of different mutant genes of the target protein is obtained by genetic engineering methods, 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 it possible to select mutants with the best features. For this purpose, for example, the genes of the desired protein or its parts are integrated into the genome of the phage - into the composition of the gene encoding the protein located on the surface of the phage particle. At the same time, each individual phage carries its own mutant protein, which has certain properties, according to which selection is made. Mutant genes are produced by "mixing" a set of genes from similar natural proteins from different organisms, usually using the polymerase chain reaction method, so that each resulting mutant protein can include fragments of many of the "parent" proteins. In essence, this approach mimics the natural evolution of proteins, but only 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 the selection of the best mutant protein variants with the desired parameters. In the case of the above task - to increase the thermal stability of the protein - selection can be carried out, for example, by growing cells containing mutant genes at an elevated temperature (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 makes it possible to better understand the structural and functional organization of protein molecules and use the knowledge gained for purposeful rational design of new proteins. Further development of protein engineering makes it possible to solve many practical problems of improving natural and obtaining new proteins for the needs of medicine, agriculture, and biotechnology. In the future, it is possible to create proteins with functions unknown in 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. Vol. 1: Genetic and protein engineering.
