The field of biotechnological is quite diverse in the medicinal sector giving rise to the creating of manifold vaccines, enzymes, and proteins for saving human lives. Concerning the same, Recombinant protein expression is one of the powerful tools that is being introduced to safeguard people against numerous diseases.
In this guest blog, we will be covering a short overview of this protein and the process of making it.
Recombinant protein expression is a proven technique, utilized in laboratories, hospitals, pharmaceutical companies, and other businesses to synthesize proteins through the integration of an alien gene into a host organism. This technique has therefore transformed the synthesis of therapeutic proteins, vaccines, enzymes, and even bio-based products.
The use of recombinant DNA technology is known as recombinant protein expression. In other words, a gene of interest is inserted into an expression vector and the resulting vector is transduced into a host cell to form the protein encoded in the gene of interest. This process is useful in research on the function of proteins, in the development of drugs, and in the manufacturing of enzymes for use in industries.
Recombinant proteins can be expressed in their host systems which include bacterial; yeast, insect, and mammalian systems partly determined by the complexity of the proteins and the general results that will be expected. For instance, human therapeutic proteins such as insulin and monoclonal antibodies are expressed in the mammalian system because human proteins require competent folding and post-translational modifications, which are not provided adequately by bacteria.
Understand the Important Steps
Some of the key steps of recombinant protein expression include gene cloning, transformation, and expression and purification of proteins.
- Cloning the Gene First
The first step involves obtaining the gene of interest and inserting it into a cloning vector which is an expression vector. Among them, the vector serves as a transporter of the gene into the host cell on behalf of the transduction. Common expression vectors are; plasmids, which are circular DNA molecules.
- At first, the gene is amplified by using a process called PCR.
- Following that, the amplified gene is then inserted into a particular vector.
- Selection markers (e.g., antibiotic resistance genes) are often included in the vector to identify cells that successfully take up the vector.
- Transformation and Transfection
Transformation occurs in bacteria and yeast systems, where the cells selected take DNA from their surroundings.
- Transformation is used in mammalian and insect cells, and the main methods include lipid-mediated transfection or electroporation through which the DNA is introduced transiently into the cell membrane of the cell that takes up the foreign DNA from the environment.
- In the process of transfection, it is employed for insect cells and follows particular methods like lipid-mediated transfection which makes the cell membrane permeable to DNA.
- Selecting Clones that are Positive
Finally, after transformation or transfection, not all cells will end up expressing the recombinant DNA, therefore a need to screen for cells containing the vector of interest. This is achieved through selective makers for example resistance to antibiotic genes present in the vector. The cells are cultured on selective media where the appropriate antibiotic to the vector is added which will kill all cells that have not incorporated the vector.
- Using the Best of Protein Expression
The transformed clones are then made to produce the recombinant protein.
- At first, a bacterial expression system is used with promoters like T7 and then induced by triggering with a chemical.
- Specifically, yeast systems rely on galactose-inducible promoters.
- On the other side, insect cells depend on baculovirus-mediated expression.
Recombinant Protein Expression Applications
- Medicine: Manufacture of about drugs including insulin, growth factors, and monoclonal antibodies.
- Research: Preparation of proteins for expression structure determination, enzyme activity, and function assessment.
- Industrial: Production of enzymes employed in the cleaning industry, the manufacture of processed foods, and the production of biodiesel.
Conclusion
The large-scale production of proteins for various applications in biotechnology has always been made possible by recombinant protein expression technology. These decisions determine the efficiency of the process and production of proteins of desired quality for distinct purposes ranging from industrial enzymes to therapeutic proteins. This knowledge of how the technology works enhances the process through which both researchers and industries apply it to other fields.