The Escherichia coli expression system is a commonly used prokaryotic expression system. Its basic principle involves inserting the target gene into an expression vector and then introducing it into E. coli host cells, utilizing the cell's own protein synthesis machinery to achieve efficient production of the target protein. Generally, the expression vector contains elements such as a promoter, signal peptide sequence, multiple cloning sites, and terminator, enabling the efficient expression of the target protein in E. coli.
The Escherichia coli expression vector is crucial for introducing the target gene into bacterial cells and achieving efficient protein expression. Generally, the expression vector contains the following basic elements: promoter, signal peptide sequence, multiple cloning sites, and terminator. The promoter initiates the transcription of the target gene, the signal peptide sequence directs the target protein to be located inside or outside the bacterial cell, the multiple cloning sites allow the insertion of the target gene DNA sequence, and the terminator terminates transcription and translation. Depending on the expression needs, additional tags such as His tag, GST tag, etc., may be added for protein purification.
To achieve efficient expression of the target protein, optimization of expression conditions is necessary. This includes selecting and adjusting the inducer concentration, temperature, cultivation time, and cell density. Common inducers include IPTG and L-arabinose, and their concentrations can be optimized based on the target protein and expression vector. Additionally, temperature and cultivation time are critical factors influencing expression levels. Typically, low temperature (e.g., 25°C) and appropriate cultivation time can enhance protein solubility and expression levels. Control of cell density is also vital as excessively high or low cell density may affect protein expression efficiency.
The expressed protein usually exists in the form of inclusion bodies and needs to undergo protein purification steps to obtain the target protein. Common purification methods include affinity chromatography, ion exchange chromatography, and gel filtration chromatography. Among them, affinity chromatography is the most commonly used method, utilizing His tags or GST tags to purify the protein through binding to affinity resins. After affinity chromatography purification, the purity of the target protein can be verified using techniques like SDS-PAGE or Western blot.
In the Escherichia coli expression system, the target protein is typically present as inclusion bodies, and its proper folding and post-translational modification may be affected. To obtain biologically active proteins, protein refolding and modification are necessary. A commonly used method involves dissolving the inclusion bodies and adding reducing agents (e.g., DTT) and folding additives (e.g., L-coenzyme A) to facilitate proper protein folding. Moreover, coenzymes or other auxiliary factors can be added during the expression of recombinant proteins to achieve modifications like glycosylation and phosphorylation.
The Escherichia coli expression system finds widespread applications in basic biological and applied research. It is used for high-throughput screening, drug target discovery and validation, as well as the production of biopharmaceuticals like vaccines and growth factors. Furthermore, E. coli serves as an initial platform for expressing other complex protein expression systems, simplifying expression and purification processes for subsequent research.
Despite numerous advantages, the Escherichia coli expression system still faces challenges in expressing complex, membrane, and toxic proteins. In the future, further optimization of expression vector design, cultivation conditions, and purification methods can enhance expression efficiency and purity. Additionally, harnessing the advantages of other expression systems, such as yeast expression systems and mammalian cell expression systems, can meet diverse research needs. Overall, the Escherichia coli expression system will continue to play a significant role in protein expression and biotechnological research.
Q1: When expressing the target protein in Escherichia coli, I always get protein expression in the form of inclusion bodies. How can I solve this problem?
A1: Expression in the form of inclusion bodies is a common issue in the Escherichia coli expression system. You can try optimizing the induction conditions (such as induction temperature and induction time). Using lower induction temperature and shorter induction time may improve protein solubility.
Q2: During the Escherichia coli expression process, I find that the protein expression level is very low. How can I increase the expression level?
A2: Low expression levels can be caused by various factors. You can try using efficient expression vectors and strong promoters to increase the expression level. Additionally, selecting appropriate host strains and optimizing culture conditions are also essential for enhancing expression levels.
Q3: I always observe partial degradation of the expressed protein in Escherichia coli. How can I address this issue?
A3: Protein degradation may result from protein instability or proteolytic degradation. You can try using protein stabilizers, such as protease inhibitors, to prevent protein degradation. Furthermore, optimizing culture and expression conditions might enhance protein stability.
Q4: My expressed protein always contains impurities, making purification challenging. What can I do to solve this problem?
A4: The presence of impurities might be due to incorrect protein folding or proteolysis during expression. You can try optimizing induction conditions, such as reducing induction temperature and time, to improve proper protein folding. Additionally, using efficient purification methods like affinity chromatography or size-exclusion chromatography can help remove impurities.
Q5: The protein I expressed in Escherichia coli forms insoluble aggregates. How can I address this issue?
A5: Protein aggregation into insoluble precipitates might be caused by overexpression or improper folding. You can try reducing expression levels and using lower induction temperature and shorter induction time to minimize protein aggregation. Additionally, adding protein stabilizers and optimizing culture conditions may improve protein solubility.
Q6: The expressed protein in Escherichia coli forms insoluble precipitates. How can I solve this problem?
A6: Protein insolubility might be due to overexpression or protein instability. You can try reducing expression levels and using lower induction temperature and shorter induction time to minimize protein precipitation. Additionally, adding protein stabilizers and optimizing culture conditions may improve protein solubility.
Q7: The expressed protein in Escherichia coli tends to form oligomers. How can I address this issue?
A7: Protein oligomerization might be inherent to the protein's structure or caused by inappropriate expression conditions. You can try optimizing culture and expression conditions to reduce protein oligomerization. Additionally, using appropriate affinity chromatography or gel filtration methods can help separate protein oligomers.
Q8: The expressed protein in Escherichia coli exhibits abnormal modifications. How can I address this issue?
A8: Abnormal protein modifications might be due to specific modifying enzymes present in the host strain. You can try using host strains lacking the relevant modifying enzymes, such as BL21(DE3) pLysS. Additionally, optimizing culture and expression conditions may help reduce abnormal protein modifications.
Q9: The expressed protein in Escherichia coli shows solubility issues. How can I address this problem?
A9: Protein solubility issues might be due to protein instability or aggregation. You can try using protein stabilizers, such as protease inhibitors, to enhance protein stability. Additionally, optimizing induction conditions, such as lowering induction temperature and reducing induction time, might improve protein solubility.
Q10: The expressed protein in Escherichia coli exhibits low expression and instability. What is a comprehensive solution to this problem?
A10: Low expression and instability might result from various factors. A comprehensive solution may involve optimizing the choice of expression vector and promoter, optimizing induction conditions, using protein stabilizers, selecting appropriate host strains and culture conditions to improve expression levels and stability. Additionally, designing well-planned experiments and comparing the effects of different conditions can help find the most suitable comprehensive solution for your expression issues.
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