Recombinant proteins are proteins generated in an appropriate host system based on exogenous DNA rather than the host's native DNA. The exogenous DNA, also called the gene of interest (GOI), is introduced into the host cells through a suitable expression vector and then expressed to proteins using the host genetic machinery.
Currently, recombinant protein production is one of the most powerful techniques used in life sciences. Due to their advantages such as high purity, specificity, efficiency, safety, customization, scalability, and consistency, recombinant proteins have been widely applied in various fields, including medicine, research, biotechnology, etc.
Among protein-based biopharmaceuticals, recombinant proteins form the largest category, encompassing enzymes, hormones, cytokines, growth factors, blood clotting factors, monoclonal antibodies (mAbs), and antibody-related products (e.g., Fc-fusion proteins and antibody fragments). They are used for therapeutics, diagnostics, and drug discovery and development, as well as vaccine development and production.
With the emergence of recombinant DNA technology, many therapeutic proteins that were extracted from natural sources originally can be recombinantly produced in different expression systems including bacterial, yeast, mammalian, and plant hosts.
Therapeutic recombinant proteins can be produced by modifying or altering the DNA via genetic engineering techniques, providing important therapies for a variety of diseases, such as diabetes, cancer, infectious diseases, autoimmune diseases, and genetic disorders.
In many genetic disorders, individuals may lack functional enzymes due to genetic mutations. Recombinant enzymes can be designed to mimic the missing or defective enzymes. Recombinant glucocerebrosidase and recombinant alpha-galactosidase A are well-established enzyme replacement therapies (ERT) for Gaucher's disease and Fabry'disease, respectively.
Some genetic disorders involve deficiencies in cytokines (encompassing molecules such as interleukins, interferons, and colony-stimulating factors), which are signaling proteins regulating immune responses and cell growth. Recombinant cytokines can be administered to compensate for these deficiencies. Recombinant erythropoietin (EPO) can stimulate red blood cell production and is used in anemia treatment.
Some recombinant cytokines such as immune checkpoint proteins also play a role in immunotherapy by amplifying immune response against certain types of cancer. They function to stimulate the immune system, evoke immune cells, and enhance anti-tumor reactions.
Genetic conditions may lead to hormonal imbalances. Recombinant proteins can be engineered to mimic hormones and restore normal physiological functions. Recombinant insulin is used to control and treat diabetes. Recombinant human growth hormone (rhGH) is used in stimulating growth in children with growth disorders.
Hemophilia, a genetic disorder, involves deficiencies in blood clotting factors. Recombinant clotting factors, such as factor VIII and factor IX, are essential for blood clotting and is used in hemophilia treatment.
Recombinant antibodies, especially monoclonal antibodies (mAbs), have revolutionized the treatment of various cancers, autoimmune diseases, and infectious diseases. Rituximab is a CD20 monoclonal antibody that is widely used for lymphomas. Trastuzumab (Herceptin) and adalimumab (Humira) are used in the treatment of breast cancer and autoimmune diseases (like rheumatoid arthritis), respectively.
In immunoassays, recombinant proteins are used as antigens to identify specific antibodies in patient samples, facilitating the diagnosis of infectious diseases, autoimmune disorders, and allergies. Additionally, these proteins also serve as calibrators and controls in diagnostic assays to ensure consistency and accuracy in measuring analyte concentrations. This is critical for the reliability of diagnostic results.
Recombinant proteins enable the development of targeted therapies tailored to specific molecular targets or pathways, improving treatment outcomes while minimizing side effects. Recombinant technology allows for the customization of therapeutic proteins to match the specific genetic variations present in individual patients.
Recombinant proteins are engineered to express specific antigens derived from pathogens such as viruses or bacteria, which are highly immunogenic and capable of inducing a protective immune response. These recombinant antigens such as surface proteins, subunits, or epitopes, are key components in various vaccine platforms, including protein subunit vaccines, virus-like particle (VLP) vaccines, and conjugate vaccines.
Recombinant protein vaccines not only show a high safety profile but also are more stable in comparison to mRNA vaccines. Recombinant hepatitis B surface antigen (HBsAg) is used in hepatitis B vaccines. Human papillomavirus (HPV) vaccines include recombinant virus-like particles.
Recombinant proteins are used in high-throughput screening assays to identify potential drug candidates. Researchers can efficiently test large compound libraries against specific targets, expediting the drug discovery process.
Recombinant proteins are also used to design biological assays for studying the effects of potential drug candidates. In preclinical studies, these recombinant proteins help researchers understand the new drug's mechanism of action and assess its efficacy.
Figure 1: Applications of recombinant proteins in medicine
Recombinant proteins are essential tools in biological research, changing the way studying and understanding cellular processes such as cell signaling, metabolism, growth, replication and death, transcription, translation, and protein modification.
These proteins allow researchers to investigate the functions of specific genes and proteins in a controlled environment. Recombinant proteins serve as valuable reagents in experiments, aiding in protein purification, structural analysis, and investigation of protein-protein interactions, receptor-ligand binding, and protein signaling pathways.
Recombinant proteins have proven performance in several laboratory techniques such as ELISA, WB, and IHC. They can be used to develop enzymatic assays. When used in conjunction with a matched antibody pair, recombinant proteins can be used as standards such as ELISA standards. Moreover, recombinant proteins can be used as positive controls in WB.
Figure 2: Applications of recombinant proteins in research
Recombinant proteins are also used in industry, food production, agriculture, and biomaterials and bioengineering.
In the breeding industry, enzymes can be added to animal feed to increase the nutritional value of feed ingredients, reduce feed and waste management costs, support animal gut health, enhance animal performance, and improve the environment. Amylases are used in the food industry to increase flavor, sweetness, and texture in various food items.
Cellulases are crucial enzymes in biofuel production, particularly in the conversion of lignocellulosic biomass into biofuels such as bioethanol. Proteases play a crucial role in detergent manufacturing, contributing to the effectiveness of laundry detergents and other cleaning products.
Besides, lactic acid bacteria (LAB) have been used for a long time for the production of fermented foods. LAB has recently been engineered for the expression of recombinant proteins, which would have wide applications such as improving human/animal digestion and nutrition.
Recombinant proteins have found various applications in agriculture, for example, crops can be genetically engineered to express proteins that confer resistance to pests, diseases (caused by viruses, bacteria, or fungi), or herbicides, contributing to improvements in crop yield and quality, and overall agricultural sustainability.
Recombinant proteins are used to design and produce functional biomaterials with tailored properties such as bioactivity, biocompatibility, and structural integrity. By incorporating specific protein sequences, these materials can mimic natural extracellular matrices, promoting cell adhesion, proliferation, and tissue regeneration.
Recombinant proteins contribute to the field of tissue engineering by providing bioactive components that support cell growth and tissue development. They can be engineered to mimic the structural and signaling features of native tissues, facilitating the development of organ transplantation.
Figure 3: Applications of recombinant proteins in biotechnology
However, recombinant proteins also have limitations.
* In some cases, the production of recombinant proteins is complex, expensive, and time-consuming.
* The recombinant proteins produced in cells may not be the same as the natural forms. This difference may reduce the effectiveness of therapeutic recombinant proteins and even cause side effects. Additionally, this difference may affect the results of experiments.
* A major concern for all recombinant drugs is immunogenicity. All biotechnologically produced therapeutics may exhibit some form of immunogenicity. It is difficult to predict the safety of novel therapeutic proteins.
Here, we numerate several research of recombinant proteins from various fields.
Machado et al. designed a recombinant multiepitope protein for diagnosing Chagas disease (rTC) and assessed the discrimination of clinical forms and cross-reactivity [1]. Their findings indicate that rTC can diagnose T. cruzi-infected individuals with low cross-reactivity and high sensitivity and specificity, suggesting its potential as a valuable component in the production of immunodiagnostic kits for Chagas disease.
Haiqiang Zhang et al. generated a HER-2-targeting recombinant protein that was in tandem with HER-2-specific single-chain variable fragment (scFv), CCL19, and IL-7 (HCI fusion protein) can be stably obtained from transfected HEK-293 T cell strains.
This study confirmed that HCI fusion protein can facilitate the synergistic antitumor effect of the immune system and that its increases antitumor ability produced breakthrough effects on the tumor microenvironment (TME) [2].
Dan Liu et al. discovered that the immunogenicity of full-length recombinant TprK (rTprK) against Treponema pallidum subsp. pallidum (T. pallidum) effectively slowed lesion progression and decreased the T. pallidum burden [3].
Immunization with rTprK not only promptly elicited a robust Th1-like cellular response but also generated opsonic antibodies to increase macrophage-mediated opsonophagocytosis. This study underscored the significance of TprK as a crucial component in the development of a syphilis vaccine.
Ming-Shu Hsieh et al. demonstrated that recombinant lipidated FLIPr (rLF) alone induced strong anti-FLIPr antibody responses to counteract FLIPr-mediated inhibition of phagocytosis [4].
They also observed that the combination of rLF with inactivated Staphylococcus aureus or recombinant FLIPr protein could increase mucosal and systemic immune responses to inactivated virus or recombinant protein. These results suggest the potential of rLF as an adjuvant and a vaccine candidate to block the immune evasion function of FLIPr.
Overall, advancements in the field of biotechnology have increased and facilitated the production of recombinant proteins for various applications. Although recombinant proteins still have some drawbacks, their roles in medicine, research, and biotechnology are irreplaceable. We also look forward to seeing more progress in the treatment of various diseases with recombinant proteins.
References
[1] Machado, Juliana Martins, et al. 2023. Proof of Concept of a Novel Multiepitope Recombinant Protein for the Serodiagnosis of Patients with Chagas Disease [J]. Pathogens 12, no. 2: 312.
[2] Zhang, H., Ye, X., Wen, J. et al. Anti-HER2 scFv-CCL19-IL7 recombinant protein inhibited gastric tumor growth in vivo [J]. Sci Rep 12, 10461 (2022).
[3] Liu, D., Chen, R., Wang, YJ. et al. Insights into the protective immune response by immunization with full-length recombinant TprK protein: cellular and humoral responses [J]. npj Vaccines 8, 146 (2023).
[4] Hsieh, MS., Chen, MY., Hsu, CW. et al. Recombinant lipidated FLIPr effectively enhances mucosal and systemic immune responses for various vaccine types [J]. npj Vaccines 8, 82 (2023).
Further reading if you want to learn more about recombinant proteins:
Production of Recombinant Protein
CUSABIO's Five Expression Systems
How to choose a suitable expression vector?
How to express a protein with bioactivity?
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