The mammalian (293T) cell expression system is a commonly used mammalian cell expression system for recombinant protein expression. The 293T cells are derived from human embryonic kidney cells (Human Embryonic Kidney 293 cells) through transfection with the SV40 virus large T antigen gene, resulting in a stable cell line.
The mammalian (293T) expression vectors typically contain a promoter, a selectable marker gene, and the coding sequence of the target protein. When constructing the expression vector, one needs to choose suitable promoters and marker genes and correctly insert the coding sequence of the target protein. Additionally, the expression vector usually contains mammalian cell replication sequences and bacterial resistance genes for amplification and selection in Escherichia coli.
Optimization of expression conditions in the mammalian (293T) expression system is crucial to achieve optimal protein expression levels. Key optimization parameters include culture temperature, culture time, and culture medium composition. Optimized expression conditions can improve protein yield and purity.
After successful expression of the target protein, the next step is protein purification. Due to the high-level protein expression achieved by the mammalian (293T) expression system, protein purification is often complex. Common purification methods include affinity chromatography, gel filtration, and dialysis.
The mammalian (293T) cell expression system ensures proper protein folding and modifications, resulting in biologically active and functional proteins. Mammalian cells contain abundant endoplasmic reticulum and Golgi apparatus, allowing for complex protein folding and modifications such as glycosylation, phosphorylation, and acetylation.
The mammalian (293T) expression system finds extensive applications in biomedical research and industrial production. It can be used to express various proteins, including cytokines, antibodies, and vaccines. Researchers can obtain highly pure and biologically active proteins through the mammalian (293T) expression system for further studies on protein structure, function, and interactions.
In the industry, the mammalian (293T) expression system is widely employed in the production of pharmaceuticals and biologics. Through the mammalian (293T) expression system, efficient expression and production of large-scale proteins can be achieved, meeting the demands of drug development and biologics manufacturing.
Although the mammalian (293T) expression system offers many advantages in recombinant protein expression, it also faces challenges. One challenge is the stability and complexity of expression vectors, necessitating further optimization of vector structure and the selection of appropriate promoters. Additionally, improvements are still needed in large-scale production using the mammalian (293T) expression system.
In the future, with continuous technological advancements, we can expect more innovations in the mammalian (293T) expression system for protein expression. Through improvements in expression vectors, optimization of expression conditions, and the development of new cell lines, the mammalian (293T) expression system will become more flexible and efficient, becoming an essential tool in the field of recombinant protein expression.
Q1: I encountered cell detachment issues in the mammalian (293T) expression system. How can I address this problem?
A1: Cell detachment may be due to improper surface treatment of the culture dish. You can try coating the culture dish surface with materials like gelatin, polylysine, or collagen to promote cell attachment. Additionally, check the culture conditions to ensure proper medium preparation, suitable pH, and compliance with temperature and CO2 levels required for the cells.
Q2: I am expressing an exogenous protein in mammalian (293T) cells, but the expression level is low. How can I increase the expression level?
A2: Low expression levels may result from low transfection efficiency or inappropriate promoter selection. You can try using more efficient transfection reagents, optimize transfection conditions such as cell density and DNA concentration. Also, consider using stronger promoters or adding enhancers to boost the expression of the target gene.
Q3: My recombinant protein expressed in mammalian (293T) cells tends to aggregate intracellularly and cannot be properly secreted. How can I solve this issue?
A3: Protein aggregation might be due to abnormal protein folding or incomplete signal peptide sequence. You can try adding a complete signal peptide sequence to facilitate proper protein secretion or using secretion expression vectors to enhance protein secretion. Additionally, optimizing culture conditions and protein expression temperature might improve protein folding and secretion.
Q4: My mammalian (293T) cells exhibit cell toxicity after expressing the exogenous protein. How should I handle this?
A4: Cell toxicity may result from overexpression of the exogenous protein. Firstly, try optimizing the protein expression conditions, such as lowering the expression temperature, reducing expression time, or using a weaker promoter. Alternatively, you can consider using a dilution transfection method to reduce protein expression levels and alleviate cell toxicity.
Q5: In the mammalian (293T) expression system, I find that the exogenous protein expressed is undergoing degradation. How can I prevent protein degradation?
A5: Protein degradation might be due to protein instability or excessive activation of protein degradation pathways. You can add protein stabilizers (such as protease inhibitors) to inhibit protein degradation. Also, ensure sufficient protein folding and modification factors in the culture medium, which can sometimes enhance protein stability.
Q6: I am expressing an exogenous membrane protein in mammalian (293T) cells, but the membrane protein cannot properly localize to the cell membrane. How can I resolve this issue?
A6: Incorrect membrane protein localization might be due to incomplete signal peptide sequences or unsuitable local membrane environments. You can try adding the full-length signal peptide sequence or using different cell lines that are known to correctly localize membrane proteins, finding the cell type suitable for proper membrane protein localization.
Q7: In the mammalian (293T) expression system, my target protein is always expressed in the form of inclusion bodies, and I cannot obtain soluble protein. What is the solution?
A7: Expression of the target protein as inclusion bodies may result from abnormal protein folding or unsuitable expression conditions. You can try adjusting the expression temperature, culture medium components, and protein expression time. Sometimes, protein refolding strategies like freeze-thawing or adding chaperone proteins may also be needed.
Q8: The recombinant protein expressed in mammalian (293T) cells contains glycosylation modifications, but the level of glycosylation is low. How can I increase glycosylation levels?
A8: Low glycosylation levels might be due to limitations in the glycosylation pathway or insufficient expression of glycosyltransferases. You can try optimizing cell culture conditions, adding appropriate glycosyltransferases for co-expression to enhance glycosylation levels. Additionally, selecting a suitable cell line can also help improve protein glycosylation levels.
Q9: In the mammalian (293T) expression system, I noticed significant variation in protein expression levels between different batches. How can I ensure consistency in expression?
A9: Variation in expression consistency might be due to unstable cell states, culture conditions, or transfection efficiency. To ensure consistency in expression, try using cells from the same batch for expression, strictly control culture conditions and transfection procedures. Additionally, using stably transfected cell lines can ensure expression stability.
Q10: My recombinant protein expression efficiency is low in the mammalian (293T) expression system. How can I improve the expression level?
A10: Low expression efficiency may result from various factors. You can try optimizing transfection conditions to increase transfection efficiency, select more suitable promoters and signal peptide sequences, or use cell lines with higher expression capabilities. Additionally, consider optimizing culture conditions and protein expression temperature, and sometimes changing the expression strategy can also improve recombinant protein expression levels.
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