I、Overview of Protein Expression Systems

A protein expression system refers to a system composed of a host, exogenous gene, vector, and auxiliary components, through which the expression of exogenous genes in the host can be achieved. It generally consists of the following parts：

1、Host. The organism that expresses the protein. It can be bacteria, yeast, plant cells, animal cells, etc. Due to the different characteristics of various organisms, the types of proteins suitable for expression also vary.

2、Vector. The type of vector matches the host. Depending on the host, vectors are classified into prokaryotic (bacterial) expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors, insect expression vectors, etc. The vector contains the foreign gene fragment. Through vector mediation, the foreign gene can be expressed in the host.

3、Auxiliary components. Some expression systems also include auxiliary components that assist the vector in entering the host. For example, the baculovirus in the insect-baculovirus expression system.

II、Types of Protein Expression Systems

（1）E. coli Expression System

Among various expression systems, the earliest to be adopted for research and the most well-mastered is the Escherichia coli (E. coli) expression system. Due to its advantages, such as rapid cell proliferation, high yield, and the relative simplicity of IPTG-induced expression, the E. coli expression system has become the most commonly used system for producing recombinant proteins.

For expressing different proteins, different vectors are required. Known E. coli expression vectors can be divided into two types: non-fusion expression vectors and fusion expression vectors. Non-fusion expression involves inserting the foreign gene downstream of a strong promoter and an efficient ribosome-binding site (RBS) in the expression vector, with translation initiated from the AUG of the foreign gene’s mRNA. The expressed product is identical in sequence to the natural target protein. Fusion expression, on the other hand, involves fusing the DNA sequence of the target protein or peptide with that of another protein or peptide fragment and expressing it within the bacterial cells. Fusion expression vectors include secretory expression vectors, purification tag-fused expression vectors, surface display expression vectors, and chaperone-assisted expression vectors.

The advantages of the E. coli expression system include a well-understood genetic background, rapid reproduction, low cost, high expression levels, ease of purification for expressed products, good stability, strong resistance to contamination, and broad applicability.

（2）Yeast Expression System

As a rising foreign protein expression system, the yeast expression system is increasingly being utilized in genetic engineering due to its combination of the advantages of both prokaryotic and eukaryotic expression systems. This system enables high-level protein expression while possessing post-translational modification capabilities, making it a powerful tool for large-scale protein production.Commonly used yeast expression systems include:

1.Saccharomyces cerevisiae Expression System：Saccharomyces cerevisiae has been used in the brewing and baking industries for thousands of years and is considered a GRAS (Generally Recognized as Safe) organism that does not produce toxins. It has been approved as a safe organism by the U.S. FDA. However, S. cerevisiae is difficult to culture at high densities, has low secretion efficiency, and hardly secretes foreign proteins with a molecular weight greater than 30 kDa. Additionally, it cannot properly glycosylate expressed foreign proteins, and the C-terminus of expressed proteins is often truncated. Therefore, S. cerevisiae is generally not used as a host for recombinant protein expression.

2.Methylotrophic Yeast Expression System：The methylotrophic yeast expression system is the most widely used yeast expression system. Methylotrophic yeasts mainly include Hansenula, Pichia, and Torulopsis, with Pichia being the most commonly applied. The expression vectors in methylotrophic yeasts are integrative plasmids, which contain sequences homologous to the yeast chromosome, allowing for easier integration into the yeast genome. Most methylotrophic yeast expression vectors contain the alcohol oxidase gene (AOX1), and under the control of its promoter (PAOX1), foreign genes can be expressed. Methylotrophic yeasts are typically first cultured in a glycerol-containing medium to high density, followed by methanol induction as the carbon source to express the foreign protein, significantly improving expression yield. Using methylotrophic yeast, the production of foreign proteins can often reach gram levels. Compared to S. cerevisiae, their post-translational modifications are more similar to mammalian cells, avoiding hyperglycosylation.

Yeast is a single-celled, simple eukaryotic organism with straightforward cultivation requirements, rapid growth and reproduction rates, and the ability to withstand high hydrostatic pressure. When used for expressing genetically engineered products, it enables large-scale production, significantly reducing manufacturing costs.

（3）Insect Expression System

The insect expression system is a widely used eukaryotic expression system capable of performing post-translational modifications and processing similar to most higher eukaryotes, as well as transferring foreign proteins. The baculovirus expression system is a highly regarded eukaryotic expression system both domestically and internationally. Expression vectors constructed using the strong promoter of the polyhedrin gene from baculovirus structural genes enable effective, and often high-level, expression of many eukaryotic target genes.This system possesses the post-translational processing functions of eukaryotic expression systems, such as disulfide bond formation, glycosylation, and phosphorylation, allowing recombinant proteins to closely resemble natural proteins in both structure and function. Its maximum expression level can reach up to 50% of the total insect cell protein. It can express very large exogenous genes (up to 200 kDa) and has the ability to co-express multiple foreign genes simultaneously within the same infected insect cell. Additionally, it is safe for vertebrates.Since the viral polyhedrin protein constitutes a very high proportion of the total viral protein, many foreign genes have been efficiently expressed under the control of its strong promoter. Commonly used baculoviruses include Autographa californica nucleopolyhedrovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). The frequently used host cells are derived from Spodoptera frugiperda Sf9 cells, and the plasmids used for foreign gene expression are derived from the PUC series, containing a multiple cloning site and the polyhedrin promoter.

The main advantages of the baculovirus system include：

1. The expressed proteins possess complete biological functions, including proper folding and disulfide bond formation.

2. Capable of post-translational modifications.

3. High expression levels, reaching up to 50% of total cellular protein.

4. Can accommodate large foreign gene insertions.

5. Capable of simultaneous expression of multiple genes.The main disadvantage is that foreign protein expression is controlled by very late viral promoters, by which time the host cells begin to die due to viral infection.

（4）Mammalian expression system

Mammalian cells can express exogenous recombinant proteins through plasmid transfection or viral vector infection. Obtaining stable transfected cells via plasmid transfection can take several weeks or even months, whereas viral expression systems can rapidly infect cells, integrating the foreign gene into the viral vector within days. This method is particularly useful for detecting target proteins from a large pool of expression products. Mammalian cell expression vectors must contain control elements such as prokaryotic sequences, promoters, enhancers, selectable marker genes, terminators, and polyadenylation signals.

Based on the temporal and spatial differences in target protein expression, expression systems can be categorized into transient, stable, and inducible systems. A transient expression system refers to host cells that, after being transfected with an expression vector, are not subjected to selective culture. The vector DNA is gradually lost during cell division, resulting in short-lived expression of the target protein. The advantage of transient expression is its simplicity and short experimental cycle. A stable expression system involves vectors that, after entering host cells and undergoing selective culture, are stably maintained within the cells, leading to persistent and stable expression of the target protein. However, stable expression is relatively time-consuming and labor-intensive due to steps such as antibiotic selection or even pressure amplification. An inducible expression system refers to one where the transcription of the target gene is activated only upon induction by exogenous small molecules. The use of heterologous promoters, enhancers, and amplifiable genetic markers can enhance protein yield.

The mammalian expression system offers unique advantages in protein initiation signals, processing, secretion, and glycosylation, making it well-suited for expressing large, intact proteins. Foreign proteins produced by mammalian cells through post-translational modification exhibit significantly higher biological activity compared to those from prokaryotic expression systems or other eukaryotic systems like yeast and insect cells. These proteins more closely resemble their natural counterparts. However, this system has several drawbacks, including its complex composition, high technical demands, limited expression yield, low productivity, and the potential risk of viral infection.

（5）Plant Expression System

Plants possess the capability to express proteins originating from animals, bacteria, viruses, and plants themselves. Their advantages in large-scale cultivation, genetic expression and modification, along with inherent safety benefits, make plant-based production of foreign proteins an exceptionally promising research field. Various proteins including antibodies, enzymes, hormones, plasma proteins, and vaccines have been successfully expressed in plant leaves, stems, roots, fruits, seeds, as well as in plant cells and organs through genetic engineering approaches.However, extraction and purification remain the primary obstacles for large-scale recombinant protein production in plants. Doloressa et al. developed a novel approach by utilizing the endoplasmic reticulum and its signal peptides in protein synthesis. They successfully targeted three recombinant proteins - xylanase from thermophilic bacteria, green fluorescent protein from jellyfish, and human placental secreted alkaline phosphatase (SEAP) - to the apoplast. This achievement led to the establishment of two innovative recombinant protein expression systems: plant root secretion and leaf secretion pathways, which significantly simplified separation and purification procedures. These systems provide potential solutions for large-scale recombinant protein production in transgenic plants.Although the development of plant-based expression systems for foreign proteins started relatively late compared to other systems, plants have already demonstrated their capacity to produce various medically relevant proteins, food-grade proteins, and industrial enzymes and protein preparations.

III. Analysis of Advantages and Disadvantages of Protein Expression Systems

Prokaryotic protein expression systems represent both the most commonly used and the most cost-effective protein production platforms. Exemplified by the E. coli expression system, prokaryotic systems offer distinct advantages including well-characterized genetics, low production costs, high expression yields, and relatively straightforward purification of expressed products. However, their primary limitation lies in the lack of post-translational modification mechanisms - such as disulfide bond formation, protein glycosylation, and proper folding - resulting in lower probabilities of obtaining biologically active proteins.

The yeast expression system, represented by Pichia pastoris, combines several advantageous features: high expression levels, inducible expression, glycosylation patterns resembling higher eukaryotes, easy purification of secreted proteins, and suitability for high-density fermentation. Nevertheless, this system presents challenges including susceptibility of some protein products to degradation and unpredictable expression levels.

Mammalian and insect cell expression systems provide the closest approximation to native post-translational modification mechanisms, offering the highest probability of preserving biological activity. However, these systems typically suffer from lower expression yields, technical difficulties in establishing stable cell lines, and significantly higher production costs.

In summary, each expression system has its own advantages and disadvantages. Using the E. coli expression system allows for the rapid production of target proteins at a relatively low cost. However, the target protein is often expressed in the form of inclusion bodies, making purification difficult. Additionally, prokaryotic expression systems lack a comprehensive post-translational modification mechanism, resulting in lower biological activity of the expressed products. Yeast and insect cell expression systems offer high protein expression levels and low cost, but their post-translational modification systems are not fully identical to those of mammalian cells. Mammalian expression systems produce proteins that are closer to their natural state, but they suffer from low expression yields and complex procedures.Therefore, when selecting an expression system, various factors must be carefully considered, such as the nature of the target protein, production cost, expression level, safety, and expression timeline. With ongoing research on foreign 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 continue to play significant roles in recombinant protein production. Moreover, more advanced and optimized expression systems are expected to emerge.