Recombinant technology Concept overview
A Recombinant protein is a protein produced artificially by an expression host organism, aka expression system, that is transfected with a recombinant gene (target gene) isolated from another organism. The purpose is to produce the target gene encoded protein in large quantities for medical, research and academic uses.
History of recombinant protein technology
Early 1950s discovery of DNA plasmids directly led to the field of recombinant DNA (rDNA). Peter Lobban from Stanford University Medical School first proposed the recombinant DNA idea in the early 1970s. The first successful production of recombinant proteins is documented in 1972 and 1973 from Stanford and UCSF. Several people were awarded the Nobel Prize for contribution to the recombinant technology.
The pharmaceutical industry has been utilizing recombinant technology for producing protein drugs since the early 1980s. One of the most impactful achievements of recombinant protein technology is the production of recombinant protein insulin. Before recombinant protein insulin is made, diabetes treatment largely depended on insulin from animal sources. The recombinant insulin significantly lowered treatment cost and enabled a consistent and sufficient worldwide supply for diabetes treatment. The discovery of restriction enzymes and ligases catapulted the field to the ability to insert foreign genetic material and proliferate in a bacterial system.
What does a restriction enzyme do?
Restriction enzymes, more precisely restriction endonucleases, cleave DNA with high specificity in pre-defined cleaving patterns, making them the backbone of recombinant technology. Under the guide of their enzymatic properties, restriction endonucleases are able to cleave plasmids and insert the foreign gene of interest. Further characterization analysis is realized with the enhanced expression.
Individual restriction enzymes recognize specific sequences, referred to as restriction sequences. Restriction sequences are palindromic sequences, usually 4 to 8 base pairs long. Palindromic sequences read the same in both the upstream and downstream direction. When restriction enzymes find these restriction sequences, they cleave the covalent bond of either the pyrimidine (T and C) or purine (A and G), generating a 5’ phosphate and a 3’ hydroxyl group at the cleavage point.
This cleavage leaves behind blunt ends with no overlap,
therefore they can no longer base pair with other nucleotides. Other types of restriction endonucleases
leave sticky ends. These sticky ends are single strand overhangs that extend
beyond the cleavage site that may further bond with other nucleotides. Sticky
ends come in handy when the same restriction enzyme is used to cleave both the
fragment of interest and the vector. This cleavage will leave identical
overhangs that a ligase will bind (ligation), resulting in your recombinant
product (rDNA, protein, etc.).
There are several common expression systems for producing recombinant proteins, such as E. Coli, Yeast, Insect Cell lines such as Sf21, mammalian cells such as CHO, HEK, and human cell lines. These expression systems act as the factory for recombinant protein production, where the cellular mechanisms involved in protein production use the recombinant gene in the vector as a blueprint to mass produce the target protein.
E. Coli is the first organism, and still often the first choice, to be used as an expression system. It is easy to use, however cannot produce protein with post-translational modification (PTM). E. Coli lacks the cellular mechanisms of eukaryotic cells which are necessary for PTM of proteins. Thus, for producing simple proteins and for applications that do not require proteins to have PTM, E. Coli is a sufficient and convenient expression system. For more complex applications, more advanced expression systems are required.
A vector is a tool for manipulating DNA. It is the transport vehicle for the gene of the protein of interest. There are many types of vectors; some common vectors are plasmids and reverse transcription viruses. The DNA that encodes the protein is first synthesized in vitro, or cloned from native host DNA templates, then inserted into the vector DNA. The host organism is processed to take up vectors. Expression vectors, once inside the corresponding host cells, will be both transcribed and translated into proteins. They are engineered specifically to enhance the expression of target proteins.
Vectors to Recombinant Proteins
Vectors are known for producing high numbers of copies, as it is exceedingly efficient in self-replication. It is utilized as a carrier of foreign passenger DNA, to be introduced at the restriction enzyme’s cleavage site. Engineered vectors may have many recognition sites; >20 recognition sites are referred to collectively as Multiple Cloning Sites (MCS). There are many commercially available vector types for cloning and expression studies. Vectors are chosen for their ability to overexpress and produce high numbers of the proteins of interest from the carrier plasmid.
Just like the decision of which vector to use, the importance of the choice of appropriate biological system, often bacterial, cannot be understated. These systems are referred to as “protein factories”, as the necessity of a robust system is mandatory for overexpression and protein production.
Once cloning of the recombinant protein of interest is completed, the protein must be purified. Structural and functional studies, along with high-throughput and large-scale production, require proteins to be >95% pure.