Genetic engineering methods for producing proteins. New genetically engineered proteins based on recombinant antibodies against TNF Efimov Grigory Aleksandrovich. Study of the biological properties of the fluorescent sensor Vhh41-KTNFin vitro and in vivo

Send your good work in the knowledge base is simple. Use the form below

Students, graduate students, young scientists who use the knowledge base in their studies and work will be very grateful to you.

Posted on http://www.allbest.ru/

Course work

discipline: Agricultural biotechnology

on the topic: “Protein engineering”

Essay
Introduction
I. Protein engineering

1.1 The concept of protein engineering. History of development

II. Examples of engineered proteins

3.3 Some achievements of protein engineering.

Conclusion
Bibliography

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:

1. 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.

2. 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.

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

4. By changing the catalytic center of an enzyme, you can increase its specificity and reduce the number of unwanted side reactions

5. By increasing the protein’s resistance to enzymes that break it down, the procedure for its purification can be simplified.

6. By changing 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.

7. By changing the structure of the regulatory sections 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.

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

9. 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

1.1 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 center 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

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.

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

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 the creation of peptide libraries have led to the production of the millions of amino acid sequences required for effective screening to identify among them the 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, we will consider in more detail the results of using peptide libraries in the study of epitopes (antigenic determinants) of 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.

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 additional amino acid sequences are present.

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.

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

1. 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 a higher temperature.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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 carried out 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 mutagenesis modified

Bibliography

1. Protein engineering.

2. Protein engineering. Mysteries of genetics. /Vyacheslav Markin // Secrets, riddles, facts.

3. Protein engineering. // Great Russian Encyclopedia.

4. Protein engineering. // Chemist's Handbook 21.

5. Protein engineering and drug effectiveness.

6. Protein engineering. / A.I. Kornelyuk // Biopolymers and Cell.

7. Protein engineering will improve the effectiveness of drugs. // Popular mechanics.

8. Protein engineering. Receiving insulin. // Biofile - scientific and information magazine.

9. Biotechnology. Main directions and achievements. // Biology for applicants and teachers.

10. Bogdanov A.A., Mednikov B.M. Power over the gene / A. A. Bogdanov, B. M. Mednikov - M.: Education, 1989 - p.208

11. Genetic engineering. // Health.

12. Genes and chemists. // Genetics.

13. Glick B., Pasternak J. Molecular biotechnology. Principles and application / B. Glick, J. Pasternak. - M.: Mir, 2002.

14. Other areas of application of genetic engineering. / L.V. Timoschenko, M.V. Chubik // Medicine - news and technologies.

15. Egorova T.A., Klunova S.M., Zhivukhin E.A. Fundamentals of biotechnology. / T.A. Egorova, S.M. Klunova, E.A. Zhivukhin - M., 2003.

16. Protein engineering. // Chemistry and biotechnology.

17. Patrushev L.I. Gene expression / L.I. Patrushev - M.: Nauka, 2000. - 496 p.

18. Patrushev L.I. Artificial genetic systems. T. 1: Genetic and protein engineering. /L.I. Patrushev - M.: Nauka, 2004. - 526 p.

19. Rybchin V.N. Fundamentals of genetic engineering: Textbook for universities/V.N. Rybchin - St. Petersburg: Publishing house of St. Petersburg State Technical University, 2002. - 522 p.

20. Stepanov V.M. Molecular biology. Structures and functions of proteins. / V.M. Stepanov - M.: Higher School, 1996.

21. Biotechnology technologies: protein engineering, nanobiotechnology, biosensors and biochips. / Evgenia Ryabtseva // “Commercial Biotechnology” - online magazine.

22. Chernavsky D.S., Chernavskaya N.M. Protein is a machine. Biological macromolecular structures. / D.S. Chernavsky, N. M. Chernavskaya - M.: Moscow State University Publishing House, 1999.

23. Schultz G.E., Schirmer R.H. Principles of structural organization of proteins. / G.E. Schultz, R.H. Schirmer - M.: Mir, 1982.

24. Brannigan J.A., Wilkinson A.J. Protein engineering 20 years on // Nature Reviews. Molecular Cell Biology. 2002. Vol. 3. No. 12;

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

Posted on Allbest.ru

Similar documents

The essence and tasks of genetic engineering, the history of its development. The goals of creating genetically modified organisms. Chemical pollution as a consequence of GMOs. Obtaining human insulin as the most important achievement in the field of genetically modified organisms.

abstract, added 04/18/2013

The emergence of biotechnology. Main directions of biotechnology. Bioenergy as a branch of biotechnology. Practical achievements of biotechnology. History of genetic engineering. Goals, methods and enzymes of genetic engineering. Achievements of genetic engineering.

abstract, added 07/23/2008

Possibilities of plant genetic engineering. Creation of herbicide-resistant plants. Increasing the efficiency of photosynthesis and biological nitrogen fixation. Improving the quality of storage proteins. Environmental, medical and socio-economic risks of genetic engineering.

test, added 12/15/2011

The essence of genetic engineering, methods for identifying transgenic organisms; production and technology of GMOs, difference from traditional breeding, advantages and disadvantages. The state and prospects for the development of the market for genetically modified goods in the world.

course work, added 11/20/2010

Genetic engineering is a method of biotechnology that deals with research into the restructuring of genotypes. Possibilities of genetic engineering. Prospects for genetic engineering. Reducing the risk associated with genetic technologies.

abstract, added 09/04/2007

Genetic engineering: history of origin, general characteristics, advantages and disadvantages. Acquaintance with the latest methods of genetic engineering and their use in medicine. Development of genetic engineering in the field of livestock and poultry farming. Experiments on rats.

course work, added 07/11/2012

The sequence of genetic engineering techniques used to create genetically modified organisms. Classification of the main types of restriction enzymes used for DNA fragmentation. Enzymes that synthesize DNA on a DNA or RNA template.

presentation, added 04/27/2014

The essence of genetic and cellular engineering. The main tasks of genetic modification of plants, analysis of the harmfulness of their consumption as food. Features of hybridization of plant and animal cells. The mechanism of obtaining medicinal substances using genetic engineering.

presentation, added 01/26/2014

course work, added 05/10/2011

Fundamentals and techniques of DNA cloning. Stages of genetic engineering of bacteria. Development of genetic engineering of plants. Genetic transformation and improvement of plants using agrobacteria, sources of genes. Safety of genetically modified plants.

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 best name for this daring idea was protein engineering.

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

If you select mutants with the necessary properties, say, those 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.

Thus, we can conclude that genetic engineering has opened the way to protein engineering in its most radical form. For example, we chose a protein and wanted to replace one amino acid residue in it with another.

Before you begin replacement work, you need to prepare a DNA vector. This is viral or plasmid DNA with the gene for the protein that interests us built into it. You also need to know the nucleotide sequence of the gene and the amino acid sequence of the encoded protein. The latter is determined from the former using a genetic code table.

Using the table, it is also easy to determine what minimal changes should be made in the composition of the gene so that it begins to encode not the original, but a protein modified at our request. Let’s say that in the middle of a gene you need to replace guanine with thymine.

Because of such a small thing, there is no need to re-synthesize the entire gene. Only a small fragment of nucleotides is synthesized, complementary to the region in the middle of which the guanine nucleotide chosen for replacement is located.

The resulting fragment is mixed with a DNA vector (circular DNA), which contains the gene we need. The DNA ring and the synthesized fragment create a section of the Watson-Crick double helix. In it, the central pair is “pushed out” of the double helix, since it is formed by mutually non-complementary nucleotides.

Add four dNTPs and DNA polymerase to the solution. The latter, using a fragment stuck to a single ring, completes it to a complete ring in full accordance with the principle of complementarity.

As a result, we get almost normal vector DNA. It can be introduced into a yeast or bacterial cell for reproduction. The only thing is that this DNA differs from the original vector in a non-complementary pair. In other words, the DNA vector helix is not completely perfect.

At the very first act of doubling the resulting vector together with the bacteria carrying it, each of the daughter DNA molecules will become a perfect double helix along its entire length. However, one of the daughter molecules carries the original nucleotide pair, and the other has a mutant vector in this place, on the basis of which the mutant protein of interest to us is obtained.

Thus, protein engineering creates a mixture of cells. Some of them carry the original vector with a non-mutant gene, while other cells carry the mutant gene. It remains to select from this mixture exactly those cells in which the mutant gene is located.

Genetic engineering methods, in particular the cloning of individual genes or parts thereof, as well as DNA sequencing, have made it possible to significantly improve the methodology of mutagenesis, eliminating the main disadvantages of classical methods for inducing mutations in genomes. Classical genetic analysis involves the effect of a mutagenic factor in vivo on the entire genome, as a result of which random mutations arise in it, often multiple, which greatly complicates the identification of mutants. Mutant individuals are identified by altered phenotypic characteristics, and the nature of the mutation can be determined after DNA sequencing. Modern localized mutagenesis, in fact, involves reverse actions: first, the gene of interest or its segment is cloned, its structure is determined during sequencing, and then the required changes are made to its composition in vitro. The consequences of the induced mutation are determined after the introduction of the mutant gene into the original organism.

The simplest version of localized mutagenesis consists of treating a cloned DNA fragment with one of the mutagenic factors, but the result of such exposure will also be random changes in the structure of the fragment. More reliable and more often used methods of localized mutagenesis are carried out without the use of mutagenic factors. Among the types of mutations, deletions, insertions and nucleotide substitutions predominate.

Deletions. These types of mutations in localized mutagenesis are obtained using endonucleases. Both restriction and nonspecific endonucleases are used. The simplest case of using restriction enzymes is to cleave a genome using a restriction enzyme that introduces several breaks to form sticky ends. The resulting fragments are again closed into a ring using DNA ligase, which can lead to the formation of molecules that do not contain one of the DNA segments. This approach produces extensive deletions and is typically used in preliminary experiments to determine the function of relatively large sections of cloned DNA.

Small deletions are obtained as follows. The cloned fragment is cleaved within the vector at the appropriate site using a restriction enzyme (Fig. 21.1). The resulting linear molecule is treated with exonuclease III, which hydrolyzes one strand of DNA,

starting from the 3' end. The result is a set of molecules with single-stranded 5' tails of varying lengths. These tails are hydrolyzed by ssDNA-specific S1 nuclease, and deletions are formed in the DNA. You can also use exonuclease Bal 31, which catalyzes the degradation of both strands, starting from the ends of linear DNA molecules. The course of nucleotic reactions is regulated by varying the incubation time, temperature and enzyme concentration, inducing the formation of deletions of different lengths. The resulting deletion variants of linear DNA are often provided with linkers before cyclization so that restriction sites are present in the region of the deletion. There are other modifications of the described methods.

Insertions (insertions). To obtain insertions, cloned DNA is digested with a restriction enzyme or nonspecific endonuclease, and then the resulting fragments are ligated in the presence of the segment that is wanted to be inserted into the DNA. Most often, chemically synthesized polylinkers are used as such segments (Chapter 20).

Insertions, like deletions, can disrupt the integrity of a gene or the structure of its regulatory regions, resulting in the synthesis of a defective protein (in the case of extended deletions or frameshifts, usually inactive) or changes in the transcription process of the gene of interest. In this way, regulatory mutants are often obtained and expression vectors are constructed (Chapter 20).

Point mutations . These mutations are nucleotide substitutions. To obtain them, several approaches can be used: cytosine deamination, incorporation of nucleotide analogues, incorrect incorporation of nucleotides during gap repair, etc.

The first method is based on the fact that cytosine residues in single-stranded DNA can be deaminated to form uracil by treatment with bisulfite ions. Single-stranded regions in DNA are usually obtained near restriction sites, for example, by the action of exonuclease III. After bisulfite treatment, single-stranded gaps are filled using DNA polymerase and the ends are ligated. In sites where uridylate was formed instead of cytidylate during deamination, adenylate will occupy the complementary position, and during replication of such a molecule the GC pair will be replaced by an AT pair.

Another approach to inducing substitutions is to treat cloned DNA with a restriction enzyme in the presence of ethidium bromide, which inserts between the planes of base pairs and disrupts the structure of the duplex. As a result, only a single-strand DNA break is formed. A small gap is created at the site of the single-strand break and then repaired in the presence of DNA polymerase, dATP, dGTP, dCTP and N-4-hydroxycytosine triphosphate instead of dTTP. Hydroxycytosine triphosphate is included in the chain instead of thymidylate, but during DNA replication it pairs equally well with both adenylate and guanylate. As a result of the inclusion of guanylate after an additional round of replication, the AT→GC substitution will occur at this site (Fig. 21.2). Since in this method nucleotide replacement is carried out internally

restriction site, it becomes possible to easily distinguish between vectors with the original sequence and mutant ones. To do this, it is enough to treat them with the restriction enzyme used in the experiment: the mutant molecules will not undergo cleavage.

A similar method is based on using only three of the four possible nucleotides when filling a single-stranded gap with DNA polymerase. In most cases, the enzyme stops at the point in the molecule where the complementary nucleotide to the missing one occurs. However, occasionally the DNA polymerase makes a mistake and turns on one of the three nucleotides present. This leads to the formation of ring molecules, which contain unpaired non-complementary nitrogenous bases. When such vectors are introduced into bacterial cells, some of the molecules will undergo repair of such damage. As a result, in half of the molecules after replication the original sequence will be restored, and in the other half the mutation will be fixed. Mutant molecules can be distinguished using the method described above.

Site-specific mutagenesis. The characterized methods of localized mutagenesis differ in that the sites where mutations occur are selected randomly. At the same time, the technique of site-specific mutagenesis makes it possible to introduce mutations into a precisely defined region of the gene. This is done using synthetic (obtained by chemical synthesis) oligonucleotides with a given sequence. The method is convenient in that it does not require the presence of convenient restriction sites. The method is based on the formation of heteroduplexes between a synthetic oligonucleotide containing a mutation and complementary single-stranded DNA in the vector.

Proceed as follows. A small oligonucleotide (8-20 monomers) is synthesized, complementary to the part of the gene in which they want to obtain a mutation. One or more nucleotide substitutions are allowed in the central region of the oligonucleotide. The gene under study or its fragment is cloned as part of a vector based on the M13 phage to obtain circular single-stranded recombinant DNA. The recombinant vectors are mixed and annealed with oligonucleotides. Hybridization of the oligonucleotide with the complementary region occurs, while non-complementary nucleotides remain unpaired. The oligonucleotide acts as a primer in a polymerase reaction involving DNA polymerase in vitro. The ring is closed by ligases. The resulting circular molecule is introduced into E. coli cells, where partial repair of mutant replication sites occurs. The frequency of mutations usually varies from 1 to 50%. The selection of cells containing mutant DNA molecules can be done in several ways, among which the advantage is the method using a radioactively labeled oligonucleotide, which is used for mutagenesis. In this case, this nucleotide serves as a probe. The principle of using such a probe is based on the fact that it is completely complementary to mutant DNA and partially complementary to wild-type DNA. It is possible to select such hybridization conditions (primarily temperature) that the hybridization of the labeled probe will be stable only with the mutant DNA sequence, which can be detected by autoradiography.

The method of site-specific mutagenesis is especially valuable because it allows you to isolate mutations without controlling their phenotypic manifestation. This method opens up new opportunities for studying the functions of gene regulatory elements, allows you to change the “strength” of promoters, optimize ribosome binding sites, etc. One of the main applications of this methodology is protein engineering.

Protein engineering. This phrase denotes a set of methodological techniques that make it possible to reconstruct a protein molecule by targeted introduction of appropriate mutations into a structural gene (site-specific mutagenesis) and, consequently, the desired amino acid substitutions into the primary structure of the protein.

An illustrative example of the construction of more active proteins are the experiments of Fersht and co-workers with the enzyme tyrosyl-tRNA synthetase from the bacteria Bacillus stearothermophilus. Analysis of the consequences of amino acid substitutions in the active site of this enzyme led to the conclusion that the removal of groups that form weak hydrogen bonds with the substrate can improve its affinity for the substrate. It was found that threonine-51 (occupies position 51 in the peptide) forms a long and weak hydrogen bond with the oxygen of the ribose ring when binding tyrosyl adenylate. At the same time, it was found that proline occupies the same position in E. coli bacteria. Site-specific mutagenesis of the gene that determines the structure of B. stearothermophilus tyrosyl-tRNA synthetase made it possible to replace thr-51→pro -51 in the peptide. As a result, the binding of ATP in the active center of the enzyme sharply improved, and its catalytic activity increased 25 times.

Another, no less significant example of protein reconstruction of practical importance is the modification of subtilisin from Bacillus amyloliquefaciens, carried out by Estell et al. Subtilisins are serine proteinases secreted by bacilli into the external environment. These enzymes are produced on a large scale by the biotechnology industry and are widely used in detergents. The disadvantage of subtilisins is a sharp decrease in proteolytic activity under the influence of oxidizing agents, including those contained in washing powders. The goal of reconstructing the BPN subtilisin molecule was to stabilize it against chemical oxidation.

In preliminary experiments, it was found that in the presence of hydrogen peroxide, subtilisin quickly reduces activity due to the oxidation of the methionine-222 residue, which is converted into the corresponding sulfoxide. Using site-specific mutagenesis methods, this methionine residue was replaced with all other 19 protein amino acids. Plasmids with mutant genes were introduced into strains with deletions in the corresponding genes, and the properties of the produced subtilisins were analyzed. Mutants with serine and alanine222 turned out to be quite stable to the action of peroxide. The most active mutant was the one containing the cysteine-222 residue; its specific activity was 38% higher than that of the wild-type strain.

In a similar way, it was possible to increase the activity of b-interferon. Other achievements of protein engineering include studies in elucidating the transforming activity of oncoproteins; changing the thermostability of enzymes, for example, obtaining thermolabile renin and thermostable a-amylase; increasing the efficiency of insulin binding by the corresponding plasma membrane receptor due to the replacement of histidine with aspartate at position 10 of the b-chain of the hormone, as well as many other examples. A large number of protein engineering products have already found practical application in production processes.

1.1 The concept of protein engineering. History of development

Figure 1. Scheme of how protein engineering works.

Protein engineering

Targeted protein modification. In targeted protein modification, the scientist uses detailed knowledge of the protein's structure and function to make the desired changes. Generally, this method has the advantage...

Protein engineering

Species and speciation

Aristotle used the term "species" to describe similar animals. After the appearance of the works of D. Ray (1686) and especially C. Linnaeus (1751-- 1762), the concept of species was firmly established in biology as the main...

Higher nervous activity in adulthood

The functioning of the brain remained an unsolved mystery for humanity for many years. Not only clergy, but also scientists who professed idealism connected all mental processes in the body with a mysterious soul...

Genetic algorithms in the problem of optimization of real parameters

What is called the standard genetic algorithm was first described and explored in detail in the work of de Jongh...

Genetic Engineering

Genetic engineering appeared thanks to the work of many researchers in various branches of biochemistry and molecular genetics. For many years, proteins were considered the main class of macromolecules. There was even an assumption...

The use of genetic engineering in the treatment of diseases and the creation of medicines

Genetic engineering appeared thanks to the work of many researchers in various branches of biochemistry and molecular genetics...

History of genetics

After the widespread dissemination of the teachings of Charles Darwin, one of the first critics to point out a weak point in the theory was the Scottish researcher F. Jenkins. In 1867, he noted that in Darwin's theory there was no clarity on the question of...

Concepts for the development of modern technologies and energy

To facilitate physical labor, since ancient times, various devices, mechanisms and machines have been invented that enhance human mechanical capabilities. But only a few mechanisms helped a person to do work...

Features of cloning

Breeds of chickens and their modern distribution

Poultry farming in most countries of the world occupies a leading position among other branches of agricultural production, providing the population with highly valuable dietary food products (eggs, meat, delicious fatty liver)...

The problem of the existence of humanity in the light of Vernadsky’s theory of the noosphere

Based on observations of natural phenomena, the idea that living beings interact with the external environment and influence its changes arose a long time ago...

Cytogenetics as a science

Cytogenetics is the science of the material basis of heredity. She studies the features of the structure, reproduction, recombination, changes and functioning of the genetic structures of the cell, their distribution in mitosis...

Evolution of groups of organisms

Evolutionary theory is the doctrine of the general patterns and driving forces of the historical development of living nature. The purpose of this teaching: to identify patterns of development of the organic world for subsequent management of this process...

Protein engineering 6 A set of methods and approaches for studying proteins and obtaining proteins with new properties MAIN TASKS Create a clone library of nucleotide and amino acid sequences Investigate the effects of single substitutions of amino acid residues on protein folding and functions Develop methods for effectively modifying proteins to give them the necessary properties Develop methods and approaches for screening and selection of proteins with required properties

Rational design Rational design The need for knowledge about the spatial organization of the protein The need for knowledge about intra- and intermolecular interactions Imperfection of methods and equipment a direction aimed at creating new proteins de novo by their spatial design

Directed evolution of protein molecules is a direction aimed at creating new proteins through selection 1 obtaining a library of random amino acid sequences 2 selecting polypeptide chains that have at least a small degree of the required properties 3 using random mutagenesis obtaining new library of proteins that are used in the next round of selection or using genetically engineered constructs expressing new proteins

Directed evolution of protein molecules (options) rational redesign using directed mutagenesis replace specific amino acid residues in the active center of the enzyme engineering of protein surfaces using mutations change sections of the polypeptide chain in the vicinity of amino acid residues that are close together on the surface of the protein globule, but located at a considerable distance in the polypeptide chain apart from each other

Screening and selection of proteins with specified properties random screening improved screening selection each protein is examined for the presence of the required properties; the selection of proteins from the library occurs randomly; each protein is examined for the presence of the required properties; the selection of proteins from the library occurs randomly; it is possible if the objects that make up the library differ phenotypically (for example, in the presence of enzymatic activity); conditions are created for the selective preservation of the components of the library that have certain properties (phage, cell display); conditions are created for the selective preservation of the components of the library; which have certain properties (phage, cell display) detection of a protein with the required properties among a large number of macromolecules that make up the resulting clone library

Phage display The goal is to display foreign proteins on the surface of the phage. The method was developed in 1985 for the filamentous bacteriophage M13. (genes pIII and pVIII are suitable target sites for the insertion of a foreign cDNA fragment) The goal is to expose foreign proteins on the surface of the phage. The method was developed in 1985 for the filamentous bacteriophage M13. (genes pIII and pVIII are suitable target sites for the insertion of a foreign cDNA fragment) a hybrid gene is constructed consisting of the coding sequences of the target protein and one of the phage envelope proteins by the bacteriophage; E. coli is infected during phage assembly; the hybrid proteins are included in the phage particle

Phagmid Helper phage Phage genome Infection of E.coli with a helper phage E.coli cells transformed with a plasmid library / phagemid are infected with a helper phage to obtain phage particles on the surface of which various variants of the target protein are exposed E. coli cells transformed with a plasmid library / phagemid , are infected with a helper phage to obtain phage particles on the surface of which various variants of the target protein are exposed

Prospects for the practical use of protein engineering Medicine: *for the production of new drugs; for the creation of diagnostic tools and the production of vaccines; *for studying the mechanisms of the immune response, as well as diseases of the immune system Ecology: *for obtaining biocatalysts in the form of whole cells with enzymes immobilized on their surface; *for obtaining biosensors for diagnostics and environmental monitoring; *for the creation of bio adsorbents to remove toxic substances and heavy metal ions from the environment

Measuring glucose using an enzyme electrode (schematic representation of L. Clark's experiment). Oxidation of glucose by the enzyme glucose oxidase in the presence of oxygen: glucose + O 2 H 2 O 2 + glucono-1,5-lactone. H 2 O 2 is reduced on the platinum electrode at a potential of +700 mV; the current flowing in the circuit is proportional to the concentration of hydrogen peroxide (i.e., indirectly, glucose).