The expression system of a protein is the process by which protein is made in cells. A specific type of DNA called messenger RNA (mRNA) is transcribed from genomic DNA using enzymes known as RNA polymerases. The mRNA then leaves the nucleus via pores in the nuclear membrane and is translated into protein sequences by ribosomes.
The expression of a protein is vital for understanding and manipulating it in experiments and many other applications, like understanding how they work in living organisms and creating new ones.
For example, if you want to study how a protein functions in an organism, you first need to know its role. To do this, you need to find out where the gene coding this protein sequence lies within its genome so that you can then express or induce that gene’s expression.
This process involves extracting DNA from an organism – by isolating whole cells and extracting their DNA.
Once you have the DNA, you need to isolate where the gene of interest lies within it. Then copy (clone) it into a plasmid before transforming it into bacteria cells so that they may replicate themselves with copies of your DNA of interest. The next step is to induce expression using Isopropyl β-D-1-thiogalactopyranoside (IPTG) so that these bacteria produce proteins for your research purposes.
There are three main expression systems used by a protein production service to study proteins and determine their function in living organisms.
Baculovirus Expression System
Baculovirus Expression Vector System (BEVS) is a robust and versatile system for expressing foreign proteins. O’Reilly et al. engineered insect viruses to replace viral genes with foreign DNA. This new baculovirus vector is suitable for large-scale production in insect cells. It has opened up avenues for the expression of proteins in higher eukaryotic systems and transformed eukaryotic cells, which expand the use of this technology beyond the initial field of protein production.
If you’re not familiar with how the system works, it consists of two main components: a bacterial host and an expression vector. The expression vector is a plasmid that contains instructions for making a protein.
DNA in the vector is cut into pieces and inserted into the host bacteria, then replicated and transcribed into mRNA. This mRNA is then translated into protein. That protein is secreted into a liquid medium (which may contain nutrients), where it can be harvested to measure its levels.
The system is easy to use than other systems because you don’t have to worry about keeping cell growth rates high or inducing specific cell types for the protein expression to work.
The baculovirus expression system has been used with success for many years. It is highly efficient and reliable.
The baculovirus expression system has several significant benefits:
- It can express foreign genes with high efficiency and yield. It also has high post-translational modification ability, ensuring that the expressed protein will have better structure and function and produce functional proteins.
- The process of recombinant baculovirus preparation does not involve the use of mammalian cells, which reduces the risk of contamination with human pathogens.
Bacterial Expression Systems
The Bacterial Protein Expression System generates biologically active recombinant proteins using inexpensive bacterial host cells as protein expression vats. Bacteria such as E. coli can be transformed with DNA, which encodes your protein of interest. They will then produce the recombinant protein from the cloned DNA. The desired recombinant protein can then be purified from the bacterial homogenate and used for biochemical applications.
This is an easy and quick way to generate and isolate milligram amounts of your recombinant protein. Furthermore, it requires very little time or effort. Thus, it is ideal for laboratory experiments requiring large amounts of purified recombinant protein for assays, crystallography, or other downstream applications.
The Bacterial Protein Expression System has several benefits:
- Recombinant proteins are produced in milligram amounts in a few days
- Bacterial cell walls are removed using sonication, eliminating the need for enzymatic digestion or other lysis techniques.
- Transformed bacteria can be grown in inexpensive media to produce large quantities of recombinant protein with minimal effort.
Mammalian Cells Expression System
The mammalian cell expression system expresses proteins in a complex intracellular environment that resembles natural physiological conditions. Therefore, it is often used for proteins that cannot be produced using conventional bacterial or insect systems.
Recombinant DNA technology is used to construct expression cassettes that are transfected or infected into host cells. Expression vectors with constitutive promoters are most used in this system. But, inducible and regulatable promoters can also be utilized.
The commonly used vector systems are the lentivirus and retrovirus. However, adenovirus, herpes simplex virus (HSV), and adeno-associated virus (AAV) have been successfully employed for gene delivery into various cell types. The major advantage of viral vectors is their ability to transduce many cell types efficiently. Once inside the host cell, the recombinant gene becomes stably integrated into chromosomal DNA.
However, using viral vectors has several disadvantages, including safety concerns related to endogenous genes present in the vector backbone, low transgene copy number, and limited capacity for large transgenes due to packaging constraints. Other non-viral methods include protoplast transfection and electroporation. But they are limited by low efficiencies and the inability to maintain stable expression of foreign genes.
In conclusion, proteins are required for all living things to survive. Protein is essential because it provides structure, stability, and function to the human body. The protein expression system includes a messenger RNA molecule transcribed from a segment of genomic DNA using enzymes. This mRNA piece then directs the protein synthesis via a process known as translation. Overall, protein expression is an integral part of everyday life, and we need to know about it!