The Importance of Recombinant Protein Technology in the Field of Biomedicine

Recombinant protein is a protein synthesized in vitro by genetic engineering technology. This technology uses molecular biology methods to insert the target gene into an expression vector of a host cell, so that the host cell can efficiently express the gene and produce the corresponding protein. The host cell can be Escherichia coli, yeast, insect cells or mammalian cells. The production process of recombinant protein includes gene cloning, vector construction, host cell transformation, protein expression and purification.

Recombinant protein technology has many advantages. First, it can produce a large amount of high-purity protein to meet the needs of research and application. Secondly, through recombinant protein technology, mutants or fusion proteins that are difficult to obtain in natural proteins can be expressed, thereby realizing functional research. [1] In addition, recombinant protein technology is widely used in the field of medicine, such as the production of vaccines, therapeutic antibodies and enzyme preparations. Recombinant proteins are also used in structural biology research, drug screening and diagnostic reagent development. [2]

In short, recombinant protein technology achieves efficient production of target proteins through genetic engineering, which has greatly promoted the development of biomedicine and biotechnology, and plays an important role in scientific research and clinical applications. Next, we will mainly introduce its related research in the field of biomedicine.

Basic principles of recombinant protein technology

1. Gene cloning

Gene cloning is the first step in recombinant protein production, and its purpose is to extract the target gene from the donor organism and insert it into an appropriate vector. This process includes the following steps:

1.1 Extracting DNA

First, DNA is extracted from the target organism (such as humans, animals or plants). This is usually done by using a lysis buffer and an enzyme (such as proteinase K) to break the cell membrane and nuclear membrane, thereby releasing the DNA inside the cell. The extracted DNA then goes through a series of purification steps, such as phenol-chloroform extraction and ethanol precipitation, to remove impurities and obtain highly pure genomic DNA. [3]

1.2 Restriction endonuclease cleavage

The extracted genomic DNA needs to be cut into smaller fragments using restriction endonucleases. Restriction endonucleases are enzymes that can recognize a specific DNA sequence and cut DNA at that sequence. [4] These enzymes cut DNA at specific locations, producing DNA fragments with sticky ends or blunt ends. These fragments contain the target gene and can be separated by electrophoresis.

1.3 Constructing recombinant vectors

After the target gene fragment is cut by restriction endonucleases, it is inserted into an appropriate vector. Commonly used vectors include plasmids, viral vectors, and yeast artificial chromosomes. Plasmids are small circular DNA molecules that are widely used in bacterial expression systems. Viral vectors are often used for efficient gene expression in mammalian cells.

In order to insert the target gene into the vector, scientists use DNA ligase to connect the target gene fragment and the cut vector DNA together to form a complete recombinant vector. This recombinant vector usually contains regulatory elements such as promoters, terminators, and selection marker genes to ensure that the target gene can be effectively expressed in the host cell and facilitate the screening of positive clones. [5]

1.4 Transformation of host cells

The constructed recombinant vector is introduced into the host cell through transformation methods. Common transformation methods include electroporation, [6] chemical transformation, and viral infection. The electroporation method uses electric shock to temporarily electroporate the cell membrane to allow exogenous DNA to enter the cell. The chemical transformation method uses calcium ions or other chemical reagents to treat the cell to enhance its ability to absorb DNA. Viral infection uses the natural infection mechanism of the virus to deliver exogenous DNA into the host cell. [7]

2. Gene expression

Once the target gene is successfully introduced into the host cell, the gene expression process begins. In this process, the target gene is transcribed into mRNA and translated into protein. Different host systems have their own specific gene expression regulation mechanisms. Choosing an appropriate host system is the key to ensuring efficient expression of the target protein. The following are several commonly used gene expression systems and their characteristics:

2.1 Bacterial expression system

Escherichia coli is the most commonly used bacterial expression system. Its advantages include fast growth, low cost, easy large-scale culture and simple genetic manipulation. However, E. coli lacks the post-translational modification capabilities required by eukaryotic cells, such as glycosylation, which may affect the function and activity of some eukaryotic proteins. [8]

The main steps of the E. coli expression system include:

Construct a recombinant plasmid containing the target gene and introduce it into E. coli.

Induce the expression of the target gene by an inducer (such as IPTG).

Collect the bacteria and obtain the recombinant protein by cell crushing and purification. [9]

2.2 Yeast expression system

Yeast is a eukaryotic system widely used for protein expression, with similar post-translational modification capabilities as eukaryotic organisms. The yeast expression system is suitable for expressing eukaryotic proteins that require complex post-translational modifications.

The main steps of the yeast expression system include:

Construct a recombinant plasmid or yeast artificial chromosome containing the target gene and introduce it into yeast cells. [10]

Induce the expression of the target gene by changing the culture conditions or using a specific inducer.

Collect the yeast cells and obtain the recombinant protein through cell disruption and purification steps. [11]

2.3 Mammalian cell expression system

The mammalian cell expression system is the best choice for producing recombinant proteins that require complex post-translational modifications. This system is capable of complex post-translational modifications including glycosylation, phosphorylation, etc., to produce proteins with natural structure and function. However, the cost of the mammalian cell expression system is high and the culture conditions are more stringent. [12]

The main steps of the mammalian cell expression system include:

Construct a recombinant plasmid or viral vector containing the target gene and introduce it into mammalian cells by transfection or infection.

Use specific culture media and conditions to induce the expression of the target gene. [13]

Collect the culture supernatant or cells and obtain the recombinant protein through purification steps.

3. Protein purification

After the target protein is successfully expressed, a series of purification steps are required to remove host cell proteins and other impurities to obtain high-purity recombinant proteins. The main steps of protein purification include:

3.1 Cell disruption

Cell disruption is the first step in protein purification. For bacteria and yeast cells, commonly used disruption methods include ultrasonic disruption, bead milling, and high-pressure disruption. For mammalian cells, commonly used methods include osmotic disruption and freeze-thaw cycles.

3.2 Preliminary separation

The preliminary separation step includes the use of centrifugation and filtration techniques to remove cell debris and other macromolecular impurities. This step can usually significantly concentrate the target protein and remove a large amount of non-specific proteins. [14]

3.3 Affinity chromatography

Affinity chromatography is an efficient protein purification method that achieves purification through the specific binding of the target protein to a specific ligand. Common affinity chromatography methods include:

Ni-NTA affinity chromatography: used to purify recombinant proteins with His tags. The His tag specifically binds to the nickel ions on the Ni-NTA resin to achieve efficient purification of proteins. [15]

GST affinity chromatography: used to purify recombinant proteins with glutathione S-transferase (GST) tags. The GST tag specifically binds to the glutathione resin to achieve efficient purification of proteins.

Immunoaffinity chromatography: purify the target protein using the specific binding of antibodies to antigens. This method is suitable for proteins that are not tagged or difficult to tag.

3.4 Ion exchange chromatography

Ion exchange chromatography separates proteins based on their surface charges. By changing the pH or ionic strength of the buffer, the target protein can be separated from other impurity proteins. Commonly used ion exchange resins include cation exchange resins (such as CM-Sepharose) and anion exchange resins (such as DEAE-Sepharose). [16]

3.5 Gel filtration chromatography

Gel filtration chromatography (also known as molecular sieve chromatography) separates proteins based on their molecular size. Large molecular proteins pass through the pores of the gel particles first, while small molecular proteins pass through later. [17] This method is often used in the last step of purification to remove small molecular impurities and further purify the target protein.

4. Quality testing

The purified recombinant protein needs to be quality tested to ensure its purity and activity. Commonly used detection methods include:

SDS-PAGE: Analyze the molecular weight and purity of the protein by polyacrylamide gel electrophoresis.

Western Blot: Verify the expression and purity of the protein by specifically identifying the target protein with antibodies.

Mass spectrometry: Used to determine the molecular weight, amino acid sequence, and post-translational modifications of the protein.

Enzyme activity assay: For recombinant proteins with enzyme activity, their activity is measured by the conversion rate of a specific substrate. [18]

Through the above steps, scientists are able to efficiently produce and purify a variety of recombinant proteins. These proteins play a vital role in basic research, drug development, and industrial applications.

Application of recombinant protein technology in biopharmaceuticals

1.Therapeutic protein drugs

Therapeutic protein drugs are one of the most important applications of recombinant protein technology. These drugs are used to treat a variety of diseases, including diabetes, cancer, hemophilia, and autoimmune diseases. The following are some examples of major therapeutic protein drugs:

1.1 Recombinant human insulin

Recombinant human insulin is the first recombinant protein drug approved for clinical treatment. Insulin is an important hormone in the human body that regulates blood sugar levels. For diabetic patients, the supplementation of exogenous insulin is essential. Traditionally, insulin is extracted from animal pancreas, which has problems such as low purity, allergic reactions and immune rejection. Through genetic recombination technology, scientists are able to express and purify human insulin in Escherichia coli to produce safer and purer recombinant human insulin. This significantly reduces the risk of allergic reactions and immune rejection in patients and improves the therapeutic effect. [19]

1.2 Recombinant interferon

Interferon is a class of proteins with antiviral, antitumor and immunomodulatory effects. Recombinant interferon plays an important role in the treatment of chronic hepatitis B, hepatitis C and certain types of cancer. For example, recombinant interferon α-2b is widely used to treat chronic hepatitis B and hepatitis C. It controls infection by inhibiting viral replication and enhancing immune response. [20] The production of recombinant interferon ensures the high efficiency and safety of the drug by inserting the human interferon gene into an appropriate expression system for expression and purification.

1.3 Monoclonal Antibodies

Monoclonal antibodies are another important class of therapeutic protein drugs. They are able to specifically bind to target molecules, block their function or mark them for clearance by the immune system. Monoclonal antibodies show great potential in the treatment of cancer, autoimmune diseases and infectious diseases. Through genetic engineering technology, highly efficient and high-purity recombinant monoclonal antibodies can be produced. For example, trastuzumab is a monoclonal antibody used to treat HER2-positive breast cancer. It prevents cancer cell growth and proliferation by specifically binding to HER2 receptors. [21]

2.Vaccine production

Recombinant protein technology is also widely used in vaccine development and production. Traditional vaccines usually rely on the cultivation and inactivation of pathogens, which poses certain safety risks. Recombinant protein vaccines avoid direct contact with pathogens by expressing and purifying the antigenic proteins of pathogens, thereby improving the safety and stability of vaccines.

2.1 Recombinant hepatitis B vaccine

Recombinant hepatitis B vaccine is one of the examples of successful application of recombinant protein technology. Hepatitis B is a serious liver infection caused by hepatitis B virus (HBV). By inserting the hepatitis B virus surface antigen gene into yeast cells for expression and purification, a highly immunogenic hepatitis B vaccine is produced. [22] The vaccine can induce the human body to produce protective antibodies, thereby preventing hepatitis B virus infection. Recombinant hepatitis B vaccines are widely used worldwide and have significantly reduced the incidence and spread of hepatitis B.

2.2 Novel coronavirus vaccine

During the COVID-19 pandemic, recombinant protein technology played an important role. A variety of COVID-19 vaccines have been developed using recombinant protein technology, including spike protein vaccines and nanoparticle vaccines. These vaccines provide protection by expressing and purifying the key antigenic proteins of the new coronavirus to induce an immune response in the human body. [23] For example, the NVX-CoV2373 vaccine developed by Novavax is a nanoparticle vaccine based on a recombinant spike protein. It enhances immunogenicity and protective effects by expressing and purifying the coronavirus spike protein in insect cells and assembling it into nanoparticle form.

3.Gene therapy

Gene therapy is a method of treating genetic diseases by correcting or replacing defective genes. Recombinant protein technology also has important applications in gene therapy, especially in gene delivery and expression. [24]

3.1 Adeno-associated virus vector

Adeno-associated virus (AAV) is a commonly used gene therapy vector. AAV vectors have the advantages of a wide range of infection, no obvious immune response, and high gene delivery efficiency. Through recombinant protein technology, high-purity AAV vectors can be produced to carry therapeutic genes into patient cells. For example, in the treatment of inherited retinal diseases, AAV vectors are used to deliver functional genes to restore patients' vision. This method has also shown good results in the treatment of other genetic diseases such as spinal muscular atrophy.

3.2 Gene editing technology

Gene editing technology, such as CRISPR-Cas9, also relies on recombinant protein technology. The Cas9 protein in the CRISPR-Cas9 system is expressed and purified by gene recombination technology for targeted cutting and modification of specific genes. CRISPR-Cas9 technology can accurately perform specific DNA editing in the genome and has broad application prospects. For example, in the treatment of sickle cell anemia, CRISPR-Cas9 is used to correct the mutated gene that causes the disease, thereby restoring normal hemoglobin function.

4.Other Application Fields

In addition to the above main applications, recombinant protein technology also plays an important role in many other biopharmaceutical fields.

4.1 Drug Screening and Target Validation

Recombinant proteins are widely used in drug screening and target validation. By expressing and purifying target proteins, high-throughput screening can be performed to find small molecule drugs with therapeutic potential. For example, recombinant enzyme proteins are used to screen enzyme inhibitors, and recombinant receptor proteins are used to screen receptor antagonists or agonists. These efficient screening methods accelerate the discovery and development process of new drugs. [25]

4.2 Diagnostic Reagents

Recombinant proteins also play an important role in the development of diagnostic reagents. By expressing and purifying specific antigens or antibodies, diagnostic reagents for disease detection can be prepared. For example, recombinant antigen proteins are used to produce ELISA kits, and recombinant antibodies are used to produce lateral flow immunochromatography kits. These diagnostic reagents play a key role in clinical testing and disease monitoring. [26]

4.3 Biosensors

Biosensors are devices that use biological molecules to recognize specific substances and are widely used in environmental monitoring, food safety, and medical testing. Recombinant proteins play an important role in the development of biosensors. For example, recombinant enzyme proteins can be used as recognition elements of biosensors to detect the presence and concentration of specific substrates. Through genetic engineering technology, [27] recombinant proteins with high specificity and high sensitivity can be designed and produced, significantly improving the performance of biosensors.

Recombinant protein technology is of great significance in biopharmaceuticals. It is widely used in therapeutic protein drugs, vaccine production, gene therapy and other fields, significantly improving the effect of disease treatment and prevention. With the continuous advancement of technology, [28] recombinant protein technology will face more opportunities and challenges in future development. By improving protein production efficiency, optimizing expression systems, developing new purification technologies and expanding application areas, recombinant protein technology will play a more important role in biopharmaceuticals and other biotechnology fields.

Future development direction of recombinant protein technology

Recombinant protein technology has made significant progress in the fields of biopharmaceuticals and biotechnology, but with the continuous development of science and technology, there are still many aspects that need to be further optimized and improved. The following are several major directions for the future development of recombinant protein technology. [29]

1.Improving protein production efficiency

Improving the production efficiency of recombinant proteins is an important way to reduce costs and increase production. The main challenges faced by current recombinant protein production include low expression levels, protein degradation, and insufficient post-translational modifications. In order to improve production efficiency, scientists are exploring a variety of strategies.

1.1 Optimizing gene sequences

By optimizing the coding sequence of the target gene, the expression level of the protein can be significantly improved. Gene optimization includes adjusting the frequency of codon usage, removing mRNA secondary structure, and increasing the accessibility of ribosome binding sites. Using computer-aided design tools, scientists can design gene sequences that are more suitable for the host expression system, thereby increasing the expression of proteins. [30]

1.2 Modifying host cells

Modifying host cells is another important way to improve protein production efficiency. Through genetic engineering, the protein synthesis capacity and stability of host cells can be enhanced. For example, in Escherichia coli, the expression and stability of recombinant proteins can be increased by overexpressing chaperone proteins and inhibiting protein degradation pathways. In mammalian cells, recombinant proteins with natural functions can be produced by optimizing post-translational modification pathways. [31]

1.3 Improving culture conditions

Optimizing culture conditions is also an important strategy to improve protein production efficiency. The growth rate of host cells and the level of protein expression can be significantly improved by optimizing the culture medium composition, regulating the culture temperature and pH value, etc. In addition, the use of high-density cell culture and bioreactor technology can further improve the yield and quality of proteins.

2.Optimize the expression system

Different expression systems have different advantages and disadvantages. Future development will focus on optimizing and improving existing expression systems and developing new expression systems to meet the production needs of different proteins.

2.1 Bacterial expression system

Bacterial expression systems, especially Escherichia coli systems, are widely used because of their low cost and high expression. However, the disadvantage of bacterial systems is that they lack the post-translational modification functions required by eukaryotic organisms. Future development directions include:

Develop bacterial expression systems that can perform post-translational modifications, for example, by introducing glycosylation enzyme systems to achieve protein glycosylation modification.

Improve expression vectors, enhance the activity of promoters and enhancers, and increase protein expression.

Optimize fermentation processes to increase cell density and protein yield.

2.2 Yeast expression system

Yeast expression systems are capable of relatively complex post-translational modifications and are an effective way to produce eukaryotic proteins. Future improvement directions include:

Develop new yeast species and expression systems, such as Pichia pastoris and Hansenula polymorpha, to increase protein expression and modification levels.

Gene editing technology is used to transform yeast cells to enhance their protein synthesis and secretion capabilities.

Optimize fermentation processes and purification processes to improve protein production efficiency and purity.

2.3 Mammalian cell expression system

Mammalian cell expression systems are capable of complex post-translational modifications and are the best choice for producing recombinant proteins with natural functions. Future development directions include:

Improving mammalian cell lines, for example, by stably expressing key modification enzymes through genetic engineering to improve the level of protein modification.

Developing new expression vectors and regulatory elements to increase protein expression and stability. [32]

Optimizing culture medium components and culture conditions to reduce cell apoptosis and increase protein production.

2.4 New expression systems

In addition to traditional expression systems, scientists are also exploring new expression systems to meet the production needs of different proteins. For example, plant expression systems: inserting target genes into plant cells through genetic engineering, and using the growth and secretion capabilities of plants to produce recombinant proteins. Plant expression systems have the advantages of low cost and large production scale, and are suitable for large-scale production of vaccines and therapeutic proteins.

3.Develop new purification technologies

Purification technology is the key to obtaining high-purity recombinant proteins. Future development will focus on developing more efficient and low-cost new purification technologies.

3.1 Nanomaterial purification technology

Nanomaterials have high specific surface area and excellent physical and chemical properties, and can be used to develop new purification technologies. For example:

Magnetic nanoparticles: By combining the target protein with magnetic nanoparticles, rapid and efficient separation and purification can be achieved. The magnetic nanoparticle purification technology is simple to operate, has a high recovery rate, and is suitable for large-scale production. [33]

Functionalized nanomaterials: By modifying the surface of nanomaterials and giving them specific affinity and selectivity, efficient purification of target proteins can be achieved. For example, protein purification is performed using materials such as functionalized carbon nanotubes and graphene.

3.2 Microfluidic purification technology

Microfluidic technology uses tiny channels and precisely controlled liquid flow to achieve efficient protein separation and purification. Microfluidic purification technology has the following advantages:

Efficient separation: Efficient protein separation can be achieved by precisely controlling the flow rate and liquid phase conditions.

Automation and continuity: Microfluidics systems can achieve fully automated operation, suitable for continuous production, and significantly improve production efficiency and consistency.

Small sample volume: Microfluidics technology requires a small sample volume and is suitable for proteins that are expensive or difficult to prepare in large quantities.

3.3 High-efficiency affinity chromatography technology

Affinity chromatography is an important method for protein purification. Future development will focus on improving the selectivity and efficiency of affinity chromatography. For example:

New affinity ligands: Develop new affinity ligands with high specificity and high affinity, such as artificial antibodies and nucleic acid aptamers designed using protein engineering technology. [34]

Multimodal chromatography technology: Combine affinity chromatography with other separation methods (such as ion exchange and hydrophobic interaction) to achieve multi-dimensional separation and improve purification efficiency and purity.

4.Expanding application areas

The application areas of recombinant protein technology are constantly expanding, and will involve more biomedical and industrial fields in the future.

4.1 New therapeutic proteins

In addition to traditional therapeutic protein drugs, recombinant protein technology will also be used to develop new therapeutic proteins. For example:

Gene editing tools: such as the CRISPR-Cas9 system, which produces efficient Cas9 protein through recombinant protein technology for gene therapy and gene function research.

Protein drug carriers: Develop drug carrier systems based on recombinant proteins to achieve targeted delivery and controlled release of drugs, and improve therapeutic effects and safety.

4.2 Diagnostic reagents and biosensors

Recombinant proteins play an important role in the development of diagnostic reagents and biosensors. Future development directions include:

High-sensitivity diagnostic reagents: Use recombinant proteins to develop highly sensitive and highly specific diagnostic reagents to improve early disease detection and monitoring capabilities.

Multifunctional biosensors: Develop multifunctional biosensors through recombinant protein technology to achieve simultaneous detection of multiple biomarkers, which are used in environmental monitoring, food safety, medical testing and other fields. [35]

4.3 Industrial enzymes and biocatalysts

Recombinant protein technology has a wide range of applications in the development of industrial enzymes and biocatalysts. For example:

High-efficiency industrial enzymes: Optimize the activity, stability and specificity of enzymes through genetic engineering to develop high-efficiency industrial enzymes for food processing, textiles, pharmaceuticals and environmental protection.

Biocatalysts: Use recombinant protein technology to develop high-efficiency biocatalysts for chemical synthesis and biotransformation, replace traditional chemical catalysts, and reduce environmental pollution and energy consumption.

Summary

Recombinant protein technology is of great significance in the field of biomedicine. Through genetic engineering, this technology can efficiently produce high-purity target proteins to meet the needs of research and application, and can express mutants or fusion proteins that are difficult to obtain from natural proteins to achieve functional research. Recombinant protein technology is widely used in the production of vaccines, therapeutic antibodies and enzyme preparations, and plays an important role in structural biology research, drug screening and diagnostic reagent development. Recombinant protein technology achieves efficient production of target proteins through genetic engineering, which has greatly promoted the development of biomedicine and biotechnology and plays an important role in scientific research and clinical applications.

In the future, the development of recombinant protein technology will face more opportunities and challenges. In order to further improve production efficiency, optimize expression systems, develop new purification technologies and expand application areas, scientists will continue to explore a variety of strategies. By optimizing gene sequences, transforming host cells, improving culture conditions and developing new expression systems, the production efficiency and quality of recombinant proteins will continue to improve. At the same time, the development of new purification technologies such as nanomaterials and microfluidics will further improve the efficiency and cost-effectiveness of protein purification. The application areas of recombinant protein technology will also continue to expand, including new therapeutic proteins, diagnostic reagents and biosensors, which will bring more innovation and development to the biomedical and industrial fields.

In short, the importance of recombinant protein technology in biomedicine cannot be ignored. With the continuous advancement of technology, recombinant protein technology will continue to play a key role in disease treatment and prevention, gene therapy, diagnosis and industrial applications, promote the overall progress of biotechnology, and provide new solutions to global health and environmental problems.

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