What Is Protein Engineering?

Protein engineering is the study of protein chemistry, protein crystallography, and protein dynamics to obtain information about the physical and chemical properties and molecular properties of proteins. Based on this, the purposeful design and transformation of the genes encoding proteins is performed through genetic engineering techniques. Obtain a transgenic biological system that can express proteins. This biological system can be a transgenic microorganism, a transgenic plant, a transgenic animal, or even a cellular system ^[1] .

Chinese name: Protein engineering
Foundation: Genetic Engineering

Applied discipline: Protein chemistry, etc.
Features: Modifying proteins

Protein engineering concepts

Based on the structural laws of protein molecules and the relationship between their biological functions, use chemical, physical, and molecular biological methods to modify or synthesize existing proteins, or to create a new protein to meet human needs Demand for production and life.

Detailed protein engineering

Protein is the material basis and the only form of all life activities. It is also the material basis or medicine for diagnosing and treating diseases. The number of human proteins not only far exceeds the number of genes, but because of the variability and diversity of proteins, protein research technology is far more complicated and difficult than nucleic acid technology. Therefore, human protein constitutes the most important research content in the post-genomic era, and has infinite and broad research prospects.

Protein is the embodiment of life. Without protein, life will no longer exist. However, some of the natural proteins found in living organisms are often unsatisfactory and need to be modified. Because proteins are made up of many amino acids connected in a certain order, each protein has its own unique amino acid sequence, so changing the key amino acids in it can change the properties of the protein. The amino acid is determined by the triplet code, so long as you change one or two bases that make up the genetic code, you can change the protein. An important way of protein engineering is to redesign the gene responsible for encoding a certain protein according to people's needs, so that the synthesized protein becomes more in line with human needs. This technique of modifying the molecular structure of a protein by causing site-directed mutations of one or several bases is called gene site-directed mutation technology.

Protein engineering is an emerging research developed on the basis of genetic recombination technology, biochemistry, molecular biology, molecular genetics and other disciplines. field. Its content is mainly

Principle of Engineering and Technology Two aspects: synthesize proteins with specific amino acid sequences and spatial structures as needed; determine the relationship between protein chemical composition, spatial structure, and biological function. Based on this, it is one of the most fundamental goals of protein engineering to realize the prediction of the spatial structure and biological function of proteins from amino acid sequences, and design and synthesis of new proteins with specific biological functions.

There is no uniform definition of protein engineering. It is generally believed that protein engineering is to change or design and synthesize proteins with specific biological functions through genetic recombination technology. In fact, protein engineering includes the isolation and purification of proteins, the analysis, design and prediction of protein structure and function, and the transformation or creation of proteins through genetic recombination or other means. In a broad sense, protein engineering is the use of physical, chemical, biological, and genetic recombination technologies to transform proteins or design and synthesize new proteins with specific functions. By means of genetic engineering, mutations are introduced into the amino acid sequence of the target protein, thereby changing the spatial structure of the target protein, and ultimately achieving the purpose of improving its function. Traditional protein engineering methods mostly use random mutations to modify the target protein. With the rapid development of computer technology and bioinformatics technology, computer simulation is increasingly used in protein engineering, which leads to semi-rational design and rationalization Design and other new methods of protein engineering. For example, domestic Shangke Biomedical Shanghai Co., Ltd. rationalizes the enzyme design by shuffling protein DNA and directed evolution to improve enzyme activity. ^[2]

Protein engineering research content

Protein structure analysis

--basis

2. Structural and functional design and prediction

Basic Application and Verification

3. Create and / or modify proteins-new proteins

End goal

Basic approaches to protein engineering

Protein Engineering Flow Chart Starting from the expected protein function designing the expected protein structure inferring the expected amino acid sequence finding the corresponding ribonucleotide sequence (RNA) finding the corresponding deoxyribonucleotide sequence (DNA) ^[3] .

Protein engineering is based on the structural laws of protein molecules and their relationship with biological functions. Through genetic modification or gene synthesis, the existing protein is transformed, or a new protein is manufactured to meet the needs of human production and life. demand. In other words, protein engineering is the second generation of genetic engineering based on genetic engineering, and it is a multi-disciplinary field of comprehensive science and technology engineering.

Protein Engineering Structure Analysis

One of the core contents of protein engineering is to collect a large amount of information about the molecular structure of proteins in order to establish a database of the relationship between structure and function, and lay the foundation for theoretical research on the relationship between protein structure and function. The determination of three-dimensional space structure is a necessary means to verify the hypothesis of protein design, that is, the new structure has changed the original biological function. Crystallographic technology has made great progress in determining the structure of proteins, but the most obvious disadvantage is the need to isolate a sufficient amount of pure protein (a few milligrams to several tens of milligrams), prepare single crystals, and then perform complicated data collection, Calculation and analysis.

In addition, the crystal state of a protein is not the same as the natural state. This issue must be considered when analyzing. NMR technology can analyze the peptide chain structure in the liquid state. This method bypasses the difficulties of crystallization and X-ray diffraction imaging analysis and directly analyzes the structure of proteins in the natural state. Modern nuclear magnetic resonance technology has developed from one-dimensional to three-dimensional. With the aid of computers, the spatial structure of proteins, the binding of proteins to prosthetic groups and substrates, and the dynamic mechanism of enzyme catalysis can be effectively analyzed and directly simulated. In a sense, nuclear magnetic resonance can analyze protein mutations more effectively. Many foreign research institutions are working to study the combination of proteins with nucleic acids, enzyme inhibitors and proteins in order to develop highly specific medicinal proteins.

Structural and functional design and prediction

Based on the data in the database established for the analysis of the structure and function of natural proteins, the spatial structure and biological function of a certain amino acid sequence peptide chain can be predicted; on the contrary, the amino acid sequence and spatial structure of a protein can be designed based on specific biological functions. Through experiments such as genetic recombination, you can directly investigate and analyze the relationship between structure and function; you can also use molecular dynamics and molecular thermodynamics to analyze and calculate the three-dimensional structure of protein molecules based on the basic principles of lowest energy and no two atoms in the same position. And biological functions. Although work in this area is still in its infancy, it is foreseeable that a complete set of theories can be established in the future to explain the relationship between structure and function to design and predict the structure and function of proteins.

Protein engineering innovation

There are many ways to transform proteins, from simple physical and chemical methods to complex genetic recombination. Physical and chemical methods: denaturing and renaturing proteins, modifying functional groups of protein side chains, segmenting peptide chains, changing the surface charge distribution to promote the formation of certain stereo conformations, etc .; biochemical methods: using proteinases to selectively segment proteins , Use transglycosidase, esterase, acylase, etc. to remove or link different chemical groups, use transaminases to make proteins gel and so on. The above methods can only act on the same or similar groups or chemical bonds. They lack specificity and cannot work on specific sites. Using genetic recombination technology or artificially synthesized DNA, not only can the protein be modified, but also a completely new protein can be synthesized from scratch.

A protein is a peptide made up of different amino acids connected by peptide bonds in a certain order. The amino acid sequence is the primary structure of a protein, which determines the spatial structure and biological function of the protein. The amino acid sequence is determined by the DNA sequence of the gene that synthesizes the protein. Changing the DNA sequence can change the amino acid sequence of the protein and achieve the regulated biosynthesis of the protein. Before determining the relationship between gene sequence or amino acid sequence and protein function, random mutagenesis should be used to cause the deletion, insertion or substitution of base pairs, so that the research target can be limited to a certain region, thereby greatly reducing gene analysis. length. Once the target DNA is clear, you can use techniques such as site-directed mutation to conduct research.

The amino acid in a mutated protein is determined by the triple code in the gene. Just change one or two of them to change the amino acid. Usually it is to change the amino acid at a certain position to study the structure, stability or catalytic properties of the protein. The life cycle of bacteriophage M13 has two phases. The genome of bacteriophage M13 is single-stranded. After invading the host cell, it exists in double-stranded form through replication. The gene to be researched was inserted into the vector M13, a single-stranded template was prepared, an oligonucleotide (containing one or several unpaired bases) was artificially synthesized as a primer, the corresponding complementary strand was synthesized, and a closed loop was ligated with T4 ligase Double-stranded molecules. After transfection of E. coli, the double-stranded molecules were replicated in the cell, so two types of plaques were obtained, and those with mismatched bases were mutant. Then transfer to the appropriate expression system to synthesize mutant protein.

Cassette mutation A genetic modification technology proposed by Wells in 1985, cassette mutation, can produce mutants of 20 different amino acids at one site at a time, and can perform "saturation" analysis of important amino acids in protein molecules. Positioning mutations were used to create two original vectors and endogenous enzyme sites on both sides of the amino acid code to be modified. The endonuclease was used to digest the genes, and then the synthesized double-stranded DNA fragments with different changes were used to replace the digested ones. part. In this way, multiple mutant genes can be obtained in one treatment.

PCR technology DNA polymerase chain reaction is the most widely used gene amplification technology. Using the research gene as a template, using artificially synthesized oligonucleotides (containing one or several non-complementary bases) as primers, and directly carrying out the gene amplification reaction, a mutant gene will be generated. After the mutant gene is isolated, the mutant protein is synthesized in a suitable expression system. This method is straightforward, fast, and efficient.

High mutation rate technology is a time-consuming and laborious task to screen out mutants from a large number of wild-type backgrounds. There are two new mutation methods with a higher mutation rate: thio-negative chain method: the modification of the phosphate between the nucleotides of the phosphate group by sulfur (- (S) -dCTP) to certain endonucleases Tolerance, synthesis of negative strand in the presence of primers and (- (S) -dCTP), and then treatment with endonuclease, the result is only a "gap" on the positive strand, with exonuclease III from 3 ` 5` Expand the gap and exceed the mismatched nucleotides on the negative strand, and repair the positive strand under the action of polymerase, you can get genes with both strands mutant; UMP positive strand method: E. coli mutant RZ1032 Lack of uracil glycosidase and UTP enzyme, M13 can use uracil (U) instead of thymine (T) to incorporate the template in this host without modification. The mutant double-strand generated by this U-containing template was used to transform normal E. coli. As a result, the positive U-containing strand was degraded by the host, while the mutant negative strand was retained and replicated.

Protein fusion transfers a part of the gene encoding one protein to another protein gene or combines fragments of different protein genes. After gene cloning and expression, a new fusion protein is generated. This method can focus the characteristics of different proteins on one protein and significantly change the characteristics of the protein. Many of the so-called "chimeric antibodies" and "humanized antibodies" that are being studied now adopt this method.

Practical applications of protein engineering

Protein engineering improves stability

Improving protein stability includes the following aspects: (1) extending the half-life of the enzyme; (2) improving the thermal stability of the enzyme; (3) extending the shelf life of medicinal proteins; (4) resisting oxidation due to important amino acids Caused loss of activity ^[3] .

Glucose isomerase (GI) is widely used in industry. In order to improve its thermal stability, Zhu Guoping et al. Determined the 138th glycine (Gly138) as the target amino acid and then used the double primer method to perform site-directed mutagenesis of the GI gene. Proly (Pro138) was used to replace Gly138, and the recombinant plasmid containing the mutant was expressed in E. coli. As a result, the mutant half-GI had twice the thermal half-life than the wild type; the optimal reaction temperature was increased by 10-12 ° C; the specific enzyme activity was the same. . According to analysis, after Pro replaces Gly138, it may be due to the introduction of a pyrrole ring, the side chain can just fill the hole near Gly138, making the protein space structure more rigid, thereby improving the thermal stability of the enzyme.

Protein engineering fusion protein

The enkephalin (Enk) N-terminal 5-peptide linear structure is the basic functional region that binds to -type receptors. Interferon (IFN) is a broad-spectrum antiviral and tumor cytokine. Li Mengfeng et al. Chemically synthesized Enk N-terminal 5-peptide coding region and linked it to human 1 type IFN gene through a linking 3 peptide coding region to express this fusion protein in E. coli. In vitro human colon adenocarcinoma cells and glioma cells were used as models. The 3H-thymidine incorporation method was used to prove that the fusion protein had significantly higher tumor cell growth inhibitory activity than pure IFN. Naloxone competition was used to block the experiment. It turns out that the increase in inhibitory activity is indeed mediated by the Enk-directed region.

Altered protein engineering activity

Usually 30 to 60 minutes after a meal, the insulin content in human blood reaches a peak, and it returns to the basic level within 120 to 180 minutes. However, the insulin preparations currently used clinically peak 120 minutes after injection for 180 to 240 minutes, which is inconsistent with human physiological conditions. Experiments show that insulin exists as a dimer at high concentrations (greater than 10-5 mol / L), and mainly at monomers at low concentrations (less than 10-9 mol / L). The principle of fast-acting insulin is to prevent insulin from forming aggregates. The structure and properties of insulin-like growth factor-I (IGF-I) have a high degree of homology and similarity in three-dimensional structure with insulin, but IGF-I does not form a dimer. The B28-B29 amino acid sequence in the B domain of IGF-I (corresponding to the insulin B chain) is reversed compared to the B28-B29 of the insulin B chain. Therefore, the insulin B chain was changed to B28Lys-B29Pro to obtain monomer fast-acting insulin. This fast-acting insulin has passed clinical trials.

Protein engineering cancer enzyme transformation

Gene therapy of cancer is divided into two aspects: drugs act on cancer cells, specifically inhibit or kill cancer cells; drugs protect normal cells from chemical drugs, and can increase the dose of chemotherapy. Herpes virus (HSV) thymine kinase (TK) can catalyze phosphorylation of thymine and other structural analogs such as GANCICLOVIR and ACYCLOVIR. GANCICLOVIR and ACYCLOVIR lack 3 'terminal hydroxyl groups, which can stop DNA synthesis and kill cancer cells. The ability of HSV-TK to catalyze GANCICLOVIR and ACYCLOVIR can be improved by genetic mutation. One was selected from a large number of random mutations. Six amino acids were replaced near the active site of the enzyme, and the catalytic capacity was increased by 43 and 20 times, respectively. O6-alkyl-guanine is the main mutagen and cytotoxin formed by the treatment of DNA with alkylating agents (including nitroso drugs for chemotherapy), so the dosage of these nitroso drugs is limited. O6-alkyl-guanine-DNA alkyltransferase O6-Alkylguanine-DNA alkyltransferase (AGT) can remove the alkyl group on guanine O6 and play a protective role. By retroviral transfection, human AGT was expressed and protected in mouse bone marrow cells. Through mutation treatment, some positively mutated AGT genes were obtained with higher activity than the wild type. After inspection, it was found that the 139th proline in a mutant gene was replaced by alanine.

Protein engineered chimeric antibodies

The immunoglobulin is Y-shaped and is composed of two heavy chains and two light chains connected to each other through disulfide bonds. Each chain can be divided into a variable region (N-terminus) and a constant region (C-terminus). The adsorption site of the antigen is in the variable region, and the adsorption site of cytotoxin or other functional factors is in the constant region. There are three parts in each variable region that are highly variable in amino acid sequence, three-dimensional structure is a loose structure (CDR) at the -sheet end, is the binding site of the antigen, and the rest is the supporting structure of the CDR. The CDR structure of different species is conserved, so that antibodies can be engineered through protein engineering.

The murine monoclonal antibody is rejected by the human immune system, and its potential therapeutic effects are not utilized. The chimeric antibody is to replace the constant region of the murine monoclonal antibody with the constant region of the human antibody, so that its immunogenicity is significantly reduced. Such as the monoclonal antibody Mab17-1A used to treat rectal colon adenocarcinoma (COLORECTALADENOCARCINOMA). Although chimeric antibodies still have problems with immunogens, several chimeric antibodies have passed clinical trials. The so-called humanized antibody is to graft the antigen adsorption region onto the human antibody, so that the foreign peptide chain on the antibody is minimized, and the immunogenicity is also minimized. However, only when the CDR is transferred to a human antibody, its antigen adsorption capacity is very small, and several framework amino acid residues must be carried in order to maintain the original adsorption force. There is a contradiction between immunogenicity and antigen adsorption. Through amino acid substitution or computer simulation analysis, the immunogenicity can be reduced as much as possible while maintaining the original adsorption force. The first clinically applied humanized antibody, CAMPATH-1H, for the treatment of lymphogranuloma and rheumatoid arthritis, although the efficacy is significant, but still more than half of the patients have an immune response. Other humanized antibodies, such as ANTI-CD33, which treat myeloid leukemia, have negligible immune responses.

Progress in Protein Engineering

At present, protein engineering is a well-developed and fast molecular engineering. This is because after protein molecule design, efficient genetic engineering can be applied to protein synthesis. The earliest protein engineering was the molecular modification of tyrosyl-t-RNA synthetase by Forsht et al. From 1982 to 1985. He measured the structure of the enzyme-substrate binding site according to XRD (X-Ray Diffraction), used localization mutation technology to change the amino acid residues bound to the substrate, and used kinetic methods to measure the activity of the obtained variant enzymes. Mechanism of action. Perry changed the Ile (3) to Cys (3) in lysozyme in 1984, and further oxidized to form Cys (3) -Cys (97) disulfide bond, which improved the thermal stability of the enzyme and significantly improved it. Application value of this food industry enzyme. In 1987, Foster changed the Asp (99) and Glu (156) on the surface of the subtilisin molecule to Lys, which resulted in the decrease of the His (64) proton pKa from 7 to 6 in the active center. Increase your vitality by 10 times. A change in the optimal pH of industrial enzymes indicates great economic benefits. Protein engineering can also change the catalytic activity, substrate specificity, oxidation resistance, thermal denaturation, and alkaline denaturation of enzymes. This shows the power of protein engineering and its bright future. The above examples are a kind of methods for protein engineering by replacing and adding or deleting key amino acid residues. The other is a "design from scratch" approach with a typical fold. In 1988, DuPont announced that it had successfully designed and synthesized an anti-parallel -helix with 73 amino residues. This shows that, as expected, the goal of folding into new proteins by designing from scratch is already achievable and reachable. The model method of predicting structure has played a significant role in laying the foundation for molecular biology. It is increasingly clear that the primary structure of proteins contains information about higher-level structures. Combining model methods and predicting advanced structures through molecular engineering has become an issue of concern.

Protein engineering brings together the latest achievements in some frontier fields of contemporary molecular biology and other disciplines. It combines the study of nucleic acid and protein binding, protein spatial structure and biological function. Protein engineering advances the study of proteins and enzymes to a new era, opening up attractive prospects for the application of proteins and enzymes in industry, agriculture and medicine. Protein engineering has created a new era of transforming and creating proteins that meet human needs in accordance with human wishes.

Prospects for protein engineering

Protein engineering brings together the latest achievements in some frontier areas of contemporary molecular biology and other disciplines. It combines the combination of nucleic acids and proteins, the spatial structure of proteins, and biological functions. Protein engineering advances the study of proteins and enzymes to a new stage, opening up attractive prospects for the application of proteins and enzymes in industry, agriculture and medicine. Protein engineering has created a new era of transforming and creating proteins that meet human needs in accordance with human wishes. Advances in protein engineering have shown attractive prospects. For example, scientists have transformed insulin into a fast-acting drug. Today, biological and materials scientists are actively exploring the application of protein engineering to microelectronics. Electronic components made by protein engineering methods have the characteristics of small size, low power consumption and high efficiency, so they have extremely broad development prospects ^[4] .