With some developments in genetic engineering and cloning, there are now several ways of expressing and isolating heterologous proteins for research. And, considerable advances in technology have helped in expressing and isolating recombinant proteins on a larger scale. But for large scale applications like enzyme, vaccine, and antibody production, the amount of protein needed is significantly high.
In such cases, you need a system to express the protein, which is easy to maintain and culture, produce large amounts of protein, and grow rapidly. Besides, mammalian proteins tend to go through various post-translational modifications. Some of the recombinant expression systems include yeast, bacteria, mammalian, and insect systems. This post discusses the recombinant expression systems.
Recombinant protein expression systems
Proteins are considered to be the most crucial biological molecules for life. These proteins do a variety of functions in an organism, such as DNA replication, catalyzing metabolic reactions, transporting molecules, and responding to stimuli.
But when it comes to analyzing the mechanism, pathway, function, and structure of these proteins, you can realize that the quantity is not enough. In this case, you need to produce high purity and quantity proteins quickly and at a low cost in a laboratory. This is where recombinant protein technology is needed.
You can consider recombinant protein as a manipulated type of protein that can be produced in several ways to generate large amounts of proteins. You can also manufacture useful commercial products and modify gene sequences using recombinant protein. You should note that recombinant DNA encodes recombinant protein, which can be cloned in a system supporting expression of the gene as well as translation of mRNA.
It’s worth remembering that the recombinant DNA which can be the cDNA sequence of the specific protein, is usually designed to be under the control of a characterized promoter. It can express the target protein in the chosen host cell to provide a high-level protein expression. And, modifying the gene using recombinant DNA technology tends to cause expression of a large quantity of protein or a mutant protein.
Choosing the right protein expression system can be crucial when it comes to recombinant protein expression. There are many factors that you need to consider, such as intended application, target protein properly, protein cost and yield. Also, there can be challenges for most protein expression projects, especially for those involving membrane protein, large protein, nuclear proteins, and many more.
Expression systems
Today, there are various expression systems you can utilize. Remember that different expression systems can have different features and applications. Some of the systems that are usually used in research and industrial include:
Bacteria expression system
The most mature expression system and the primary use is the E. coli expression system. The key method is to transfer a vector and insert into a target DNA fragment to the host cell. You can then induce protein expression by using IPTG.
The E. coli expression system is the most widely used expression system because it has the advantages of having a clear genetic background, lost cost, fast breeding, easy purification of the product, high expression, wide application range, and many more.
But this expression system also has some downsides in the prokaryotic expression system as not all proteins are soluble. Also, there can be formation of incorrectly folded proteins in cytoplasm that can form insoluble aggregates known as inclusion bodies. As a result, this can lead to problems in purification. Aside from this, the post-translational modification process associated with the prokaryotic expression system cannot be perfect and the biological activities of the expressed protein can be low.
Yeast expression system
This expression system has some advantages when it comes to both eukaryotic and prokaryotic expression systems. It is usually utilized in genetic engineering. The complete gene sequence involving Saccharomyces cerevisiae was sequenced in 1996. Its use in the bread and brewing industry has been known for many years and are regarded as generally safe creatures that are not poisonous. The FDA has also recognized it. Therefore, the proteins expressed in yeast systems don’t require many host safety requirements.
But Saccharomyces cerevisiae system is not ideal for high-density culture because it lacks strict and strong regulation promoter. Also, the expression system has a low secretion efficiency. This is especially true with most target proteins that have molecular weight of more than 30 kD as they don’t usually secrete.
People have now developed a methanol yeast expression system which is widely utilized. Some of the commonly used methanol yeasts include H Polymorpha, Pichia pastris, and Candida bodini. Among these methanol yeasts, Pichia pastoris is the most popular one.
Remember that most of these methanol yeasts have the methanolic yeast oxidase gene-1. This exogenous gene was expressed through the promoter, PAOX1. This is a strong promoter that uses glycerol or glucose as a carbon source. Hence, the expression of AOX1 gene can be increased through the control of the gene. And, using methanol yeast so that you can express exogenous production is usually up to grams.
Baculovirus expression system
This is an insect expression system that tends to utilize a eukaryotic expression system. This system can translate and modify any foreign protein just like higher eukaryotes. Today, expressing exogenous proteins in insect cells using recombinant baculovirus has become one of the most popular expression methods. You should remember that the protein level can be at least 1 ~ 500 mg / L. However, it can be restricted and affected by several factors like the oxygen supply, culture medium, and logarithmic growth.
It’s also worth remembering that baculovirus is considered to be the largest group of well-known insect viruses. Also, it’s the most studied, earliest, and applicable insect virus. Further, the baculovirus genome is regarded as a single closed and circular double-stranded DNA molecule that measures between 80 and160 kb.
The genome of baculovirus can usually be replicated and transcribed in the nuclei of an inset. DNA replication can be gathered in the baculovirus’ bark, which happens to be flexible and can keep large parts of foreign DNA. It is a suitable host for the expression of large parts of DNA.
