Advantages and Disadvantages of Various Protein Expression Systems

In the field of modern biotechnology, the research and application of recombinant proteins have become an important engine for promoting scientific progress and industrial development. Recombinant proteins are a type of protein that is artificially synthesized in vitro through genetic engineering technology. This technology enables scientists to produce a large number of target proteins for various purposes such as basic research, clinical treatment and industrial production. The emergence of recombinant protein technology has not only greatly improved the efficiency and accuracy of protein research, but also provided new possibilities for the treatment of various diseases.

Currently, commonly used protein expression systems include bacterial expression systems (such as Escherichia coli), yeast expression systems, insect cell expression systems and mammalian cell expression systems. Each system has different characteristics in terms of expression efficiency, protein post-translational modification ability, production cost and operational complexity.

This article will compare the advantages and disadvantages of these protein expression systems in detail, and provide readers with a reference for choosing a suitable expression system by analyzing the performance of different systems in terms of expression efficiency, post-translational modification, cost and application scenarios. This not only helps researchers make wise choices when designing experiments, but also provides valuable guidance for biopharmaceuticals and industrial production. Through systematic comparison and analysis, we hope to reveal the unique advantages and potential limitations of each protein expression system, thereby promoting the further development of biotechnology.

E. coli Expression System

As a common prokaryotic expression system, Escherichia coli has been widely used in the fields of biotechnology and genetic engineering. Its high efficiency and economical characteristics make it the preferred tool for protein expression in research and industrial production. However, the Escherichia coli expression system also has some limitations. [1]

The E. coli expression system has many advantages. It has an extremely high growth rate and can quickly reach a high cell density under appropriate culture conditions, thereby achieving high-yield protein expression. Compared with other expression systems, [2] E. coli can produce a large amount of recombinant protein in a short period of time. The culture medium it uses is simple and inexpensive, and the culture conditions are not harsh, so the production cost is relatively low; the genetic manipulation technology of E. coli is very mature, [3] and the standardized operation process makes the steps of gene cloning, plasmid construction and protein expression relatively simple. This provides great convenience for researchers and industrial producers. Even inexperienced researchers can quickly perform gene manipulation and protein production after training. Due to the efficiency and economy of the E. coli expression system, it is widely used in many fields such as basic research, drug development, industrial enzyme production and agriculture. [4]

Fig.1 Protein expression workflow in E. coli [5]

But it also has some disadvantages. As a prokaryote, E. coli lacks organelles such as the endoplasmic reticulum and Golgi apparatus that are unique to eukaryotic cells. This causes some complex eukaryotic proteins to be unable to fold and modify correctly in E. coli, which in turn affects their biological activity and function. This is a major limitation of the E. coli expression system, especially for proteins with complex structures. Therefore, when we need to purify some proteins with large molecular weight and complex structure, the E. coli expression system is not a suitable choice; when overexpressing certain exogenous proteins in E. coli, inclusion bodies are often formed. [6] These inclusion bodies are aggregated by misfolded proteins, which are difficult to dissolve and purify, and require complex denaturation and renaturation processes to restore the activity of the protein. This not only increases the difficulty of operation, but also may reduce the yield and quality of the protein; it also lacks many post-translational modification mechanisms of eukaryotic organisms, such as glycosylation, phosphorylation, etc. This means that some eukaryotic proteins that require specific post-translational modifications to function may not be able to achieve their natural biological activity when expressed in E. coli. Therefore, for proteins that require these modifications, researchers may need to choose other expression systems, such as yeast, insect cells, or mammalian cells. Finally, E. coli is a Gram-negative bacterium that contains lipopolysaccharides (LPS), also known as endotoxins, in its cell wall. Endotoxins can contaminate the final product during recombinant protein production, which is a serious problem especially when these proteins are used in medical or biological products. Removal of endotoxins requires additional processing steps, which increases production costs and complexity. [7]

In summary, the E. coli expression system has become the main force in protein expression due to its advantages of high efficiency, economy and ease of operation. However, its shortcomings in protein folding, inclusion body formation, post-translational modification and endotoxin contamination also limit its performance in some high-end applications.

Yeast Expression System

Yeast expression system is a powerful tool widely used in basic research and industrial production. It is a technology that uses yeast cells to produce target proteins. Commonly used yeast species include Saccharomyces cerevisiae and Pichia pastoris. Yeast cells are unicellular eukaryotic organisms with protein synthesis, modification and secretion pathways similar to those of higher eukaryotic organisms, so they can be used to produce biologically active eukaryotic proteins. [8]

As a eukaryotic organism, yeast has a cell structure and function similar to that of higher eukaryotic organisms, and is capable of post-translational modifications of proteins, such as glycosylation, phosphorylation, and disulfide bond formation. These modifications are essential for the function and stability of certain proteins. Compared with prokaryotic expression systems such as Escherichia coli, yeast expression systems can produce proteins that are closer to their natural state. Yeast expression systems can efficiently express foreign genes, especially Pichia pastoris, which is known for its high-density fermentation and strong protein secretion capabilities. Pichia pastoris can achieve high levels of protein expression under methanol induction conditions, and due to its powerful secretion system, [9] the target protein can be secreted into the culture medium, simplifying the subsequent protein purification process.The yeast expression system is relatively simple to operate, with low culture costs, fast growth rates, and short fermentation cycles, and can obtain a large amount of target protein in a relatively short period of time.Because of these advantages, the yeast expression system has become one of the most popular expression systems used by researchers. [10]

Even though the yeast expression system is widely recognized, it still has limitations. Yeast is capable of certain eukaryotic post-translational modifications, but its modification patterns are different from those of mammalian cells. For example, the glycosylation pattern of yeast is significantly different from that of mammalian cells, which may affect the function and stability of certain glycoproteins. For proteins that require specific post-translational modifications, the yeast expression system may not fully meet the requirements; although the yeast expression system can efficiently express many proteins, for some complex proteins, the expression level may not be as good as the mammalian cell expression system. For example, some multi-subunit proteins or proteins with complex structures are less efficient in yeast. [11] The yeast expression system may need to improve the expression level of the target protein by optimizing the culture conditions and gene construction; the gene transformation efficiency of yeast is relatively low, especially for large plasmids or complex gene constructions, the transformation efficiency may not be as good as that of prokaryotic systems such as Escherichia coli. This limits the scope of application of the yeast expression system to a certain extent. In order to improve the transformation efficiency, it is usually necessary to optimize the transformation method and use efficient selection markers.

As an important genetic engineering tool, yeast expression system is widely used in various fields due to its eukaryotic expression ability, efficient protein expression, economical and safe advantages. However, it also has some limitations, such as differences in protein post-translational modifications, expression level restrictions, low gene conversion efficiency and production scale restrictions. [12]

Fig.2 The major known plasma membrane and intracellular membrane ion transporters in the yeast cell. [13]

Insect Cell Expression Eystem

The insect cell expression system is a biotechnology tool commonly used to produce recombinant proteins. It has attracted much attention due to its advantages in expressing complex protein structures. This expression system usually uses Baculovirus as a vector to introduce the target gene into insect cells for protein expression. The main insect cell lines used include Sf9, [14] Sf21 and Hi5 cells. These cell lines have high protein expression capacity and are easy to culture and maintain. Next, I will introduce the basic principles, applications, advantages and disadvantages of the insect cell expression system in depth to help you better understand the importance of this technology in biopharmaceuticals and research. [15]

The insect cell expression system has the characteristics of efficient expression and can achieve efficient protein expression in a short time. The reason is that insect cells have a high infection rate and high expression level of baculovirus, which makes it an ideal choice for large-scale protein production; insect cells have protein modification mechanisms similar to mammalian cells, such as glycosylation, phosphorylation, and hydroxylation. [16] These modifications are essential for the function and stability of proteins, making proteins expressed by insect cells closer to their natural state; the insect cell expression system avoids the problem of viral contamination in mammalian cell expression systems. Baculovirus only infects insect cells and is harmless to human and mammalian cells, which improves the safety of the production process. [17]

Insect cell expression systems also have shortcomings.Although the insect cell expression system is cost-effective in some aspects, the overall production cost is still high. Insect cell culture medium, virus amplification and protein purification require a lot of resources. This makes some small laboratories unable to afford its high costs; the insect expression system also has unstable expression levels, and the expression levels of different target proteins in insect cells may vary greatly. Some proteins are expressed at low levels in insect cells, affecting production efficiency and cost-effectiveness; sometimes there are differences in modification. [18] Although insect cells have protein modification capabilities similar to mammalian cells, in some cases, there are still differences in modification patterns. This may affect the function and activity of the protein, especially in applications with strict modification requirements; finally, baculovirus vectors may have limitations in some applications. For example, when the target gene is large or there are repetitive sequences, it may become difficult to construct and stably express baculovirus vectors.

The insect cell expression system has unique advantages in protein production and biotechnology research. Its efficient protein expression ability and complex post-modification ability make it play an important role in vaccine production, gene therapy and protein research. However, it also faces challenges such as glycosylation differences and production complexity. Even so, the insect cell expression system is still the recommended choice.

Mammalian cell expression system

Mammalian cell expression system plays an important role in biotechnology and biopharmaceuticals. It is capable of complex protein synthesis and post-translational modifications and is suitable for the production of biologically active and functionally intact recombinant proteins. [19] Mammalian cell expression system refers to the method of expressing foreign proteins in mammalian cells. These systems usually use engineered mammalian cell lines such as HEK293 (human embryonic kidney cells) and CHO (Chinese hamster ovary cells). These cell lines are widely used to produce therapeutic proteins, vaccines and proteins for research purposes. [20]

Fig.3 Illustration of a typical process to develop a mammalian cell line for recombinant protein manufacturing. [21]

Mammalian cell expression systems also have some disadvantages. The cost of mammalian cell culture is relatively high, including culture medium, gene transfection reagents and equipment. This makes the large-scale production of therapeutic proteins costly. [22] Compared with expression systems such as bacteria and yeast, mammalian cells grow slowly and have low protein production per unit time. This limits their application in some high-demand products. Mammalian cells are sensitive to culture conditions and require strict control of temperature, pH and oxygen concentration. This increases the complexity and cost of the culture process. [23]

As an important biotechnology tool, mammalian cell expression system has many unique advantages, especially in the production of complex, biologically active proteins. Despite the challenges of high cost and low yield, these problems are gradually being solved with the continuous advancement of technology. If you have a sufficient budget and time, mammalian cell expression system will be your first choice.

Summary

The selection of protein expression system is a crucial part in scientific research and industrial production. Different expression systems have their own advantages and disadvantages, so in specific applications, factors such as expression efficiency, protein post-translational modification, production cost and operational complexity should be comprehensively considered.

The E. coli expression system has become the main force in research and industrial production due to its high efficiency, economy and simple operation. However, its limitations in protein folding, inclusion body formation, post-translational modification and endotoxin contamination also limit its performance in some high-end applications. Although the yeast expression system can provide proteins closer to the native state of eukaryotic organisms, its glycosylation pattern is different from that of mammalian cells, which may affect the function and stability of some glycoproteins. The insect cell expression system is outstanding in expressing complex protein structures and providing eukaryotic post-modification, but its production cost is high and the expression level is unstable. Mammalian cell expression system is widely used in biopharmaceuticals and research because it can perform complex protein synthesis and post-translational modification, but its high cost and low yield are the main challenges.

In summary, the selection of a suitable protein expression system needs to be weighed according to the specific application requirements and the characteristics of the target protein. For basic research and industrial production that require high efficiency, economy and simple operation, the E. coli expression system is an ideal choice. For applications that require complex post-modification and high-quality protein production, yeast, insect and mammalian cell expression systems provide more suitable solutions. In the future, with the continuous advancement of technology, the performance of protein expression systems will be further improved, bringing more possibilities and innovations to scientific research and industrial production.

Reference

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