There is no doubt that recombinant protein production in microbial systems has revolutionized biochemistry. Long gone are the days when hefty kilos of plant and animal tissues or massive biological fluids volumes were required to purify only a tiny amount of a particular protein.
Now researchers embarking on new custom protein production projects requiring a purified protein needs to start thinking of a way of obtaining it in a recombinant form. It is now possible to express and purify desired recombinant proteins in large quantities, making it possible for biochemical characterization. Let’s look at the steps for custom protein production.
Gene Amplification
Gene amplification refers to the differential increase in a specific genome’s portion compared to the remaining part. That is a ubiquitous process in most organisms and has been demonstrated to occur in somatic and germline cells. The amplification of genes is mainly associated with normal developmental processes. They could also occur as sporadic genetic events with detrimental or favorable consequences to the organism. Somatic cell gene amplification has been studied in cancers affecting humans. During the development of Drosophila melanogaster, gene amplification was recognized as a physiological process. Predominantly, the mammalian cells use the mechanism of gene amplification for the overexpression of specific genes to survive under stress. For instance, when exposed to cytotoxic drugs. There has been a proposal of four models for the generation of amplifications. They include:
- Breakage-fusion-bridge cycle
- Recombination and extra replication
- Double rolling-circle replication
- Replication fork stalling
- Template switching
Insertion into Cloning Vector
It has become easy to add pieces of foreign DNA to bacteria because of modern laboratory techniques. This is done by first packaging the DNA of interest within a vector or a circular DNA molecule. Various methods are then used to induce bacteria to take up the vector. Adding foreign DNA makes the host bacterium a genetically modified and new organism.
To carry out the cloning of a stretch of DNA like a gene into a vector, you use restriction enzymes to cut out the DNA of interest and open up or expose the vector. You then add the DNA to the vector by mixing them in addition to the enzyme DNA ligase.
The process of cloning DNA into a vector involves some necessary steps, which include;
- Cut out the gene
- Open up the vector
- Stick the gene and the vector together
Sub Cloning into Expression Vector
What is subcloning? This technique re-clones a DNA fragment from one vector to another. That is useful for the easy performance of transformation, analysis, and recombination of the target genes. It is an essential tool in the toolkit of any biologist and assists in elucidating the target gene’s function and analyzing its phenotype quickly. The process of subcloning involves four steps:
- Use of subcloning technology protocol for obtaining the target fragment
In a gene library, find the target fragment and obtain a target fragment from the cDNA library. After that, the DNA is cut into several fragments bearing restriction enzymes and introduced into cells, then synthesized gene sequences in vitro.
- Connecting Enzyme Vectors and target fragments
Choose a restriction enzyme that is appropriate for the cleavage of the target fragment from the vector. This cleavage usually generates non-symmetric cohesive ends, symmetric cohesive ends, or blunt ends.
- Transformation into a host cell
Transduction and transfection are the two most common transformation methods of target fragments into cells. A recombinant phage or plasmid is transformed into a treated host cell in transfection. In transduction, on the other hand, a host cell is transducted using a virus harboring exogenous DNA. Generally, transduction turns out as more efficient than transformation.
- Identification and Screening
Vectors bearing the recognizable genetic markets are utilized to separate and distinguish the cells transformed with recombinant DNA.
Transformation into a protein-expressing host
There are various hosts for expressing proteins. They include;
Yeast Protein Expression Systems (Saccharomyces cerevisiae)
- cerevisiae comes with the benefits of highly developed genetic systems, reduced costs, and time input. That has, in turn, made it an attractive organism for the production and expression of recombinant proteins. Another advantage of yeast is that it can carry plasmids specifically designed, which is a valuable ability in an expression system of recombinant proteins.
Insect Cell Expression Systems – SF21 and SF9
Sf9 and Sf21 are cell lines derived from Spodoptera frugiperda and are frequently used as recombinant protein expression systems.
Expression Systems of Mammalian Cells – HEK293 and CHO
Mammalian cells for recombinant protein expression come with the main challenge of reduced efficiency and the levels of the expressed proteins. However, cell lines like CHO and HEK293 have been developed as efficient and transient expression systems.
Recombinant protein test for identification – Fluorescence or Western Blot
Given that commercial antibodies are available for all, you can detect tagged recombinant proteins through Western blot alongside the expression trials. That is necessary and helpful if the desired protein levels are not high enough to see SDS-PAGE. The steps that follow are the large-scale production, then isolation and purification.
Final Thought
Theoretically, the needed steps for obtaining recombinant proteins are pretty straightforward. Take the gene of interest, clone it with your desired expression vector, transform it to your choice’s host, and induce, and the protein is now ready for characterization and purification.