What are Recombinant Proteins?
What are recombinant proteins?
The synthesis of proteins via the utilization of recombinant DNA technology has been one of the most ground-breaking discoveries in scientific research in recent years. Before this, the only way to produce proteins was by isolating them from their native source. Nowadays, DNA sequences that encode a protein of interest can be inserted in to a vector and introduced into an expression system, such as mammalian cells, bacteria, yeast, or insect cells, where it can be easily expressed in and purified from. This has allowed some of the most disruptive and progressive findings to be made in proteomics research.
Recombinant proteins are proteins that have been created via the genetic recombination of DNA molecules/sequences of interest. The genetic material brought together may be from multiple (two or more) biological sources, and are most often sequences that would otherwise not be found in association with one another. It is these sequences that, when replicated and expressed, form a novel protein with known function.
Figure: Schematic representation of the synthesis of Antibody Genie Recombinant Proteins
How are recombinant proteins made?
Recombinant proteins are made through genetic engineering, also called gene splicing or recombinant DNA technology. By putting human, animal or plant genes into the genetic material of host cell expression systems these microorganisms can be used as factories or producers to make proteins for medical, academic and research uses.
For DNA to be manipulated it must be placed within a “transport vehicle” in which proteins may be produced from the genetic code of the DNA. Some of the most common host cells used for recombinant protein synthesis include; mammalian cells, bacteria, yeast cells, or insect cells.
Isolation of gene/s of interest
The synthesis of recombinant proteins begins with the identification of the genes of interest; the gene(s) that will encode the protein of interest. To isolate these genes from the native genome, DNA is treated with restriction enzymes known as endonucleases. These enzymes “cut” the gene of interest out of the genome preparing it for the cloning steps.
Amplification of gene/s of interest
Cloning is the process by which the gene(s) of interest are then inserted into an expression vector and expressed. Before this occurs however, the fragment of interest must be amplified. This step is necessary as the genetic material of interest must be in a large enough quantity for the subsequent sequencing steps. This is often carried out by polymerase chain reaction (PCR). PCR is the most common in vitro method of cloning. However, cloning may also be carried out in an in vivo setting. The most common in vivo method of cloning is done within the bacterium E.Coli.
Following amplification, the vectors must then be introduced into their host cell. Vectors may already be in their host cells if amplification is carried out in an in vivo setting. However, if replication is carried out in vitro, the host cells are prepared for the uptake of genetic material, either via mechanical (e.g. electroporation, microinjection) or chemical (e.g. calcium phosphate, heat shock) transfection methods.
Gene Selection and Expression
Significant amounts of recombinant protein are produced by the host only when expression genes are added. The protein’s expression depends on the genes which surround the DNA of interest. This collection of genes act as signals which provide instructions for the transcription and translation of the DNA of interest by the cell. These signals include the promoter, ribosome binding site, and terminator.
The piece of DNA that is inserted into the vector often has other important non-coding regions which have specific functions. These regions allows scientists to distinguish which cells have incorporated the recombinant genetic molecules, as well as allow the gene to be expressed. For example, the antibiotic-resistance gene allows the host cell to survive in its residing medium that is antibiotic-rich. Those host cells which have not taken up the vector as a result do not express the gene which is necessary for resistance to antibiotics, and thus the organism dies.
The vector often contains a tag in addition to the specific DNA sequence; this facilitates the purification of the recombinant protein. This is often added at the N-terminal end of the amino acid sequence. One of the most common tags used in recombinant DNA technology is the hexahistidine tag (His-Tag). This is a sequence of 6 histidine residues that act as a metal binding site for recombinant protein purification and expression. The His-Tag contains a cleavage site for a specific protease. His-Tag recombinant proteins are purified by Metal Chelate Affinity Chromatography such as nickel ion columns that are used as the heavy metal ion and the His-Tag protein is eluted from the metal-chelate column with Histidine or imidazole. Then the purified His-Tag protein is treated with the specific protease to cleave off the His-Tag or not if the tag doesn’t affect the active site of the protein.
The resultant product is the fully formed recombinant protein. Amplification not only occurs during the PCR step but also at this stage, as host cells have the potential to produce thousands of proteins once the vector is uptaken.
Functions of recombinant proteins
Recombinant proteins function according to the protein they have been created to resemble. Proteins may have several different functions. This depends on the subgroup which the protein belongs to. Some subgroups of proteins include:
- Antibodies (e.g. IgG, IgA, IgD)
- Hormones (e.g. follicle-stimulating hormone (e.g. FSH, Oestrogen, Serotonin)
- Enzymes (e.g. Trypsin, Thrombin Lysozyme)
- Signalling peptides (e.g. cytokines)
- Structural proteins (e.g. Titin, Ubiquitin, Laminin)
- Storage proteins (e.g. Ferritin, Gliadin)
- Transport proteins (e.g. carrier and channel proteins)
Advantages of recombinant proteins
- Allows scientists to recapitulate the endogenous activity of key biological proteins of living organisms in an in vitro setting,