In the past several years, the expression of a wide range of recombinant proteins has expanded its use particularly in screening and therapeutic applications (Andersen & Krummen, 2002). Recombinant proteins began to be expressed in different host organisms since the introduction of DNA recombinant technology in the 1970s (Demain & Vaishnav, 2009).
The production of recombinant proteins requires an appropriate expression system that will generate sufficient quantities of the protein (Brondyk, 2009). The initial step in producing recombinant proteins is cloning the required DNA and the protein is amplified in a suitable expression system (Demain & Vaishnav, 2009).
Screening applications
Screening applications usually need small quantities of recombinant proteins whereas therapeutic applications require a high volume of production that involves complex glycoproteins and antibodies (Andersen & Krummen, 2009). A variety of expression systems have been continuously developed and evaluated for the production of recombinant proteins, in which the most commonly used systems are Escherichia coli, Pichia pastoris, baculovirus or insect cells, and mammalian cells (Brondyk, 2009). A number of companies offer recombinant protein expression services to produce the proteins in the most efficient way possible using comprehensive methods.
Therapeutic applications
Recombinant proteins are used in therapeutic applications including the development of new protein drugs, vaccines, antibodies, and diagnostic reagents (Nosaki & Miura, 2021). The first used recombinant protein in history dates back in 1982, when recombinant human insulin was used as treatment (Pham, 2018). After several years, the clinical use of recombinant proteins has expanded to more than 130 of such proteins that are approved by the FDA, and more than 170 are continuously being produced and used in medicine all over the world (Pham, 2018).
Through the constant advancements in the recombinant protein production, treatment of multiple diseases and disorders was made possible by yielding a high number of such proteins at lower cost (Tripathi & Shrivastava, 2019). Human insulin, albumin, human growth hormone (HGH), Factor VIII, and many more are examples of recombinant protein pharmaceuticals produced (Demain & Vaishnav, 2009).
Drug discovery
As proteins are an essential component of biopharmaceuticals accounting for over 200 protein drug products in the market, they are synthesized in a host cell to obtain appropriate quantities (Overton, 2014). In the drug discovery process, different quantities of recombinant proteins are needed at different stages. For example, when determining protein structure that are drug targets, only small quantities of recombinant proteins are required for initial drug development studies.
In contrast, larger quantities of recombinant proteins are needed for preclinical and clinical trials during the latter part of the drug development process (Overton, 2014). Furthermore, recombinant protein production has also progressed as a subject in biomedical research and biotechnology including structure and biophysical studies, functional assays, biomarkers, and in vitro and in vivo mechanistic studies (Assenberg et al., 2013).
Choice of an expression system
The choice of an expression system is crucial in the production of recombinant proteins, depending on the intended application of the recombinant protein to synthesize sufficient quantities of the protein (Brondyk, 2009). When selecting the appropriate expression system for recombinant protein synthesis, the most essential factors to consider are protein quality, functionality, production speed, and yield (Demain & Vaishnav, 2009).
- E. coli protein expression system
The use of E. coli as an expression system remains the first choice when generating significant amounts of recombinant proteins because of its capability for rapid growth and easy manipulation without high expenditures (Rosano et al., 2019). It is also important to note E. coli was the first expression system used in producing recombinant DNA (rDNA) human insulin in 1982 (Gopal & Kumar, 2013).
The E. coli protein expression system has the ability to produce proteins in a short period of time due to its fast doubling time and quick assessment time (less than a week) for recombinant gene expression (Brondyk, 2009). Aside from the characteristics of E. coli such as being easy to culture and having a very short life cycle, it is also unchallenging to be manipulated due to its well-known genetics (Gopal & Kumar, 2013). Using E. coli as an expression system is also ideal for non-glycosylated protein functional expression and for other complex eukaryotic proteins, which leads to advances in the fundamental understanding of transcription, translation, and protein folding as well as the availability of enhanced genetic tools to improve the use of E. coli (Demain & Vaishnav, 2009). E. coli protein expression services are being offered by established laboratory service providers to help scientists maximize the potential of their research.
- Yeast protein expression system
The production of recombinant proteins may also be through yeasts such as the methylotrophic yeast Pichia pastoris and Saccharomyces cerevisiae. Yeast expression systems are used when E.coli expression systems pose folding problems or glycosylation requirements that result in limited production (Demain & Vaishnav, 2009). Since yeasts are eukaryotic, they may produce post-translational modifications such as glycosylation, and different studies focus on creating yeast glycosylation patterns to imitate human cells (Overton, 2014).
Yeast is very easy to cultivate and produce huge quantities of recombinant proteins, and some of its therapeutic applications include yeast-generated protein drugs such as vaccines and insulin (Overton, 2014). S. cerevisiae was the first yeast used for recombinant protein expression and its cell-cycle dependency and metabolic impact of heterologous protein release have been studied (Andersen & Krummen, 2002; Brondyk, 2009). However, it has been observed that P. pastoris has successfully synthesized large scale recombinant proteins, which is limited when using E. coli (Cregg et al., 2000). P. pastoris was recently used in a perfusion system to produce recombinant human chitinase for clinical research, generating more than 300 mg/L/day (Andersen & Krummen, 2002). Aside from glycosylation, disulfide bonds are synthesized by P. pastoris, which is restricted in E. coli that may result in inactive or insoluble misfolded protein when using the latter expression system (Demain & Vaishnav, 2009).
- Baculovirus expression system
Another expression system that may be used in producing recombinant proteins is through baculovirus expression in insect cells, in which the Autographa california is most commonly used (Brondyk, 2009). Insect cells are capable of performing more complicated posttranslational modifications than fungi, and have the greatest machinery for folding mammalian proteins, making them ideal for producing soluble protein of mammalian origin (Demain & Vaishnav, 2009).
Meanwhile, because of their potential for appropriate protein folding, assembly, and post-translational modification, cultured mammalian cells have become the main expression system for the synthesis of recombinant proteins for therapeutic purposes (Wurm, 2004). It has also been observed that stable transfected Chinese hamster ovary (CHO) cells produce recombinant antibodies at levels of a few grams per liter (Brondyk, 2009). Mammalian cell cultures are especially valuable since proteins are frequently produced in a completely folded and glycosylated state, reducing the need to renature them (Demain & Vaishnav, 2009).
Recombinant protein expression services are offered to help clients generate their desired proteins through efficient techniques that align with their objectives. Learn more about recombinant protein expression services and benefit from a comprehensive service program for your research study.
