Recombinant DNA technology has become a staple technique in modern molecular biology, microbiology and biochemistry and enabled the biotechnology sector to grow tremendously in the past decades.
Genetic engineering of DNA molecules by using restriction enzymes and DNA ligase enzymes to recombine genetic material from different species (hence “recombinant” and “transgenic”) has been increasingly supplemented by the growing ability to perform de novo synthesis of artificial DNA segments and cloning them into the genome of suitable host cells like Escherichia coli (E. coli) and CHO cells, thus leading to previously unbelievable applications of recombinant DNA molecules.
In this text, we want to highlight five formidable examples of such groundbreaking utilizations of rDNA technology and the resulting recombinant proteins.
Insulin is a protein hormone that is produced in specific pancreatic cells and is involved in the regulation of blood sugar levels. Some forms of diabetes are caused by the inability to produce sufficient amounts of insulin and are fatal if left untreated.
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For decades, these patients had to rely on injections of insulin from animal sources which have the disadvantages of impurities, immunogenicity and inflammation and price/supply issues.
Although Frederick Sanger elucidated the DNA sequence (nucleotide sequence) of human insulin in the 1950ies, it took another 20 years until the biochemical techniques of manipulating nucleic acids, restriction endonucleases and plasmids were sufficiently refined to reprogram bacterial cells to perform gene expression of foreign DNA and produce human insulin in pharmaceutical quality and industrial scale.
This process was impeded by the fact that insulin consists of two protein chains that are linked by disulfide bonds, meaning that two pieces of DNA must go through the processes of transcription into mRNA, subsequent translation into two polypeptides and concluding fusion of both chains.
Moreover, recombinant insulin from bacteria lacks post-translational glycosylation which has a large impact on the pharmacokinetics of the product. The whole process had to be transferred into more complex organisms like yeast to yield proper insulin.
During childhood, the body produces hormones to stimulate the bodily transition into adolescence and adulthood. Human growth hormone (hGH or somatotropin) is produced and stored in somatotropic cells in the pituitary gland and is responsible for triggering growth.
Childrens with growth hormone deficiency (e. g. Turner’s syndrome) are generally short for their age and exhibit slower development than their peers, e. g. reaching developmental milestones like standing and walking later.
While the causes for growth hormone deficiency are diverse, the treatment is generally hormone replacement therapy with hGH. Before expression of genes of interest in host organisms became possible, growth hormone had to be extracted from animal sources with all associated risks: contaminations, immune reactions and supply chain issues.
When the gene coding for hGH became available, the expressed genetic information would have yielded an inactive peptide: the human body synthesizes a prohormone that has to be processed by enzymes first in order to become active.
In the 1980ies, biotech companies achieved the genetic recombination of the fragment of DNA that codes for active hGH with suitable plasmids and human growth hormone became widely available.
Recombinant subunit vaccines work by presenting the immune system with certain distinct molecular parts of pathogens (“subunits”), thus avoiding exposure to the pathogens themselves and being very safe.
The subunits are selected to be potent antigens and lead to a strong immune response. They are combined with adjuvants and then injected into the patients in order to raise antibodies and confer immunity or a mild course of disease.
The DNA fragments that code for the relevant subunit are modified by in vitro chemical biology techniques such as polymerase chain reaction (PCR), replication and reverse transcription of RNA into DNA to optimize their expression properties.
They are transferred into host organisms that are easy to culture, such as yeast cells, and then grown in culture media. When exponential growth ceases, the culture is subjected to downstream processing, i. e. removal of cell fragments and purification of the proteins of interest.
The first recombinant subunit vaccine was developed in the 1980ies against hepatitis B and one of the recently developed ones works against COVID-19.
Gene therapies aim at genetic diseases that are caused by mutations in chromosome regions that render the production of functional protein impossible. The idea is to introduce intact copies of the respective gene into the defunct cells to restore their functionality and alleviate the symptoms.
While the concept is easy, there are numerous obstacles to overcome in the process: the human genome is contained in the nuclei of cells, which is one of the features that distinguishes eukaryotic cells from prokaryotic ones. It is very hard to transfer nucleic acids through the cell membrane, avert enzymatic degradation in the cytosol and move them through the nuclear envelope, where they have to be inserted into the residing genetic material.
One approach is to use recombinant DNA technology to reprogram viruses to insert synthetic restorative DNA strands into cells instead of their own pathogenic genes.
Even from this high-level illustration, it should be clear that the development of gene therapies makes heavy use of recombinant DNA methodologies to solve its issues.
Enzymes are used in a variety of industries: pharmaceuticals, chemical production, biofuels, foods and beverages and a high volume commodity with an associated billion dollar market. Their optimization and production is of great interest in the biotechnology sector.
Enzymes are primary gene products, as opposed to secondary metabolites like antibiotics. This means that genetic manipulation has a direct impact for the resulting proteins or enzymes and using traditional methods of altering genes like screening, selection, mutation and conjugation were giving good results.
The advent of recombinant DNA technology allowed scientists to manipulate, optimize and recombine enzymes from different sources in test tubes at the genetic level, enhancing their activities, selectivities or substrate scope, yields etc. The use of phages to introduce the resulting genes into living organisms like bacteria and yeast cells enables the production on an industrial scale.
Read more: What is recombinant DNA used for?
While considering those five examples of great applications of rDNA, an even more worthy candidate for the pinnacle of recombinant DNA technology for protein production is the production of recombinant antibodies in mammalian cells.
Antibodies are huge, complex molecules that consist of a protein scaffold with attached sugar chains as post-translational modifications. These molecular giants can be subjected to genetic fine-tuning to optimize their binding properties, to adjust their modulatory properties of the immune system and to minimize potential immunogenicity.
They are perfectly suited for therapeutic applications in humans due to their high homogeneity, purity and quality.
Recombinant antibody production service providers like evitria have perfected the science and necessary skills for recombinant antibody production and recombinant DNA technology.
While traditional methods require about 6-12 months for the generation of antibody production hybridoma cells, recombinant antibodies made by evitria can be ready for shipping in as short as 6 weeks.