Question:
Recombinant Proteins: How Are They Made?
The Protein Man Says:
After choosing an appropriate vector and host that will satisfy the requirements of your experiment, you need to go through the following steps to create your recombinant protein.
- Preparation of vector DNA. A restriction endonuclease is used to cleave the site where the foreign DNA will be inserted.
- Preparation of DNA to be cloned. The protein of interest is extracted and any contaminants (other proteins, RNA and/or smaller molecules) present in the lysate are removed. The purified proteins are then treated with a restriction enzyme to generate fragments that can be linked with the vector.
- Production of recombinant protein. DNA from the vector and from the foreign source are mixed at appropriate concentrations and exposed to an appropriate DNA ligase to link the ends together.
- Introduction to the host organism. The recombinant DNA is then inserted into a living host organism using one of the following processes – transformation, transduction, transfection and electroporation.
- Selection of organisms containing the recombinant DNA. Since only a small fraction of the host cells will end up taking the DNA, all those that haven't taken them up will be selectively killed to make sure that only those that have the genetic marker will survive and eventually replicate.
- Screening. Colonies of transgenic cells are identified from vector DNAs that have not been inserted with recombinant sequence.
Recombinant Proteins - Some Common Applications
As mentioned earlier, this technology has far reaching applications. Here are some of them.
- Medicine. Recombinant proteins are used to produce insulin, human growth hormone (HCH, somatotropin), blood clotting factor VIII (usually administered to patients with hemophilia) and hepatitis B vaccine. In addition, this technology is also responsible for developing the methods for diagnosing HIV infections.
- Biological research. Recombinant DNA can be used in mapping genes and as reagents in laboratory experiments.
- Agriculture. The technology was used to develop Golden rice, a variety of rice that aims to reduce vitamin A deficiency. It was also used to develop insect-resistant and herbicide-resistant crops.
- Food processing. Several food additives such as chymosin, an enzyme used in the manufacture of cheese, are now being produced using this technology.