The protein expression system is similar to a sophisticated instrument that continuously delivers proteins to the outside world. The central law is the simplest one which is that the terminal is the protein that performs biological functions. In order to dig deeper into protein information and develop protein potential, the amount of protein must be guaranteed.
The Generic Pathways for Protein Synthesis
- Clone or synthesize target gene
- Vector construction
- Protein expression and purification
Overview of Protein Expression Systems
The protein expression system, consisting of the host, the foreign gene, the vector, and auxiliary components, enables the expression of the exogenous gene in the host. It generally consists of the following parts:
Host: The host is the organism that expresses proteins, which can be bacteria, yeast, plant cells, animal cells, etc. The types of suitable proteins for expression vary with the characteristics of various organisms.
Vector: The type of vector is matched to the host. According to different hosts, they are divided into prokaryotic (bacterial) expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors, insect expression vectors, etc. The vector contains foreign gene fragments. Mediated by vectors, foreign genes can be expressed in the host.
Auxiliary ingredients: Some expression systems also include auxiliary components that assist the vector to enter the host. An example is the baculovirus in the insect-baculovirus expression system.
Classification of Protein Expression Systems
Escherichia Coli Expression System
Among various protein expression systems, Escherichia coli was the earliest one to be adopted for research. The advantages of the E. coli expression system include fast propagation, low cost, high expression level, easy purification of expression products, good stability, strong anti-pollution ability, and wide application range. Its disadvantages are no post-translational modifications, inclusion bodies, and difficulty in expressing large molecular weight proteins.
Recommended expression: bacterial proteins; antigenic proteins; cytokines, enzymes.
The Yeast expression system is widely used in the field of genetic engineering due to its advantages in both prokaryotic and eukaryotic expression systems. This system can express proteins at high levels and has post-translational modification functions, so it is considered to be a powerful tool for expressing large-scale proteins. The characteristic Yeast expressed by Yeast is a single-celled lower eukaryotic organism whose advantages are economical, rapid and high-yield, and partial post-translational modification; its disadvantages are non-human glycosylation and high mannose modification.
Recommended expressions: cytokines; small molecule proteins; enzymes.
Baculovirus–Insect Cell Expression System
Insect expression systems have similar post-translational modification processing and the ability to transfer foreign proteins to most higher eukaryotes. The insect baculovirus expression system is a highly respected eukaryotic expression system at home and abroad. The expression vector constructed by using the strong promoter of the polyhedrin protein in the baculovirus structural gene can enable effective or even higher expression of many eukaryotic target genes. The advantages of insect cell expression are:
- Histones with intact biological functions, such as correct protein folding and disulfide bond matching
- Protein post-translational processing modification
- High expression level, up to 50% of the total protein
- Accommodates large molecule inserts
- Expresses multiple genes simultaneously
The main disadvantage is that the expression of foreign proteins is under the regulation of very late viral promoters when the cell death has started due to viral infection.
Mammalian cells can be used to express exogenous recombinant proteins by utilizing plasmid transfection and viral vector infection. It takes weeks or even months to obtain stable transfected cells using plasmid transfection. The virus expression system allows rapid infection of cells and integration of the foreign genes into viral vectors within a few days, so it is especially suitable for detecting target proteins from a large number of expression products. Mammalian cell expression vectors must contain control elements such as prokaryotic sequences, promoters, enhancers, selectable marker genes, terminators, and polynucleotide signals.
Expression systems can be classified into transient, stable, and inducible expression systems based on the temporal and spatial differences in the expression of the target protein. The transient expression system means that host cells are introduced into the expression vector without selective culture, the vector DNA is gradually lost with cell division, and the expression time limit of the target protein is short. The advantages of the transient expression system are simplicity and a short experimental cycle. The stable expression system means that the vector enters the host cell and is selectively cultured, the vector DNA is stably present in the cell, and the expression of the target protein is durable and stable. Due to the steps such as resistance selection and even pressurized amplification, stable expression is relatively time-consuming and labor-intensive. The inducible expression system means that the transcription of the target gene is induced by exogenous small molecules before it is opened. Protein production can be increased by using heterologous promoters, enhancers, and amplifiable genetic markers.
Mammalian expression systems are uniquely suited for expressing intact macromolecular proteins in terms of protein initiation signals, processing, secretion, and glycosylation. The exogenous proteins produced by post-translational processing and modification of mammalian cells are far superior to prokaryotic expression systems and eukaryotic expression systems such as yeast and insect cells in terms of activity. And these proteins are closer to natural proteins, but the disadvantages of this expression system are complex composition, high operational technical requirements, small expression yield, low yield, and sometimes causing virus infection.
In conclusion, each protein expression system has its advantages and disadvantages. The E. coli expression system can obtain the expression product in a relatively short time and the required cost is relatively low. However, the target protein is often expressed in the form of inclusion bodies, which makes product purification difficult; besides, the post-translational processing and modification system of the prokaryotic expression system is not perfect, and the biological activity of the expressed product is low. Yeast and insect cell expression systems have high protein expression levels and low cost, but the post-translational processing modification system is not exactly the same as that of mammals. The mammalian expression system produces proteins that are closer to the natural state, but have low expression levels and are cumbersome to operate.
Therefore, when selecting a protein expression system, various factors should be fully considered, such as the nature of the protein to be expressed, production cost, expression level, safety, and expression cycle. With the continuous research on exogenous gene expression systems and the discovery of more expression mechanisms and influencing factors, it is believed that in the near future, both prokaryotic and eukaryotic expression systems will still have a place in the production research of recombinant proteins, and more and more perfect expression systems will appear.