There are 6 basic steps for making recombinant proteins.
- Amplification of the gene of interest. This is done through PCR, in which the amplification of the gene of interest is carried out with 2 sets of primers and enzyme Taq polymerase.
- Insertion into the cloning vector. The insertion is carried out by using restriction enzymes that cut and paste DNA and subsequently produce a molecule of recombinant DNA; this process is known as transfection.
- Transformation into a protein expressing host, such as bacteria, yeast, or mammalian cells.
- Cells are harvested by centrifugation, and tests for identification of recombinant protein are carried out by using Western blot or fluorescence.
- Large-scale production of these proteins, by using prokaryotic and eukaryotic expression host systems.
- Isolation and purification of these recombinant proteins. Harvested cells are lysed to release cellular components such as recombinant DNA, and then purified to remove any remaining impurities.