Q1: If the customer provides plasmids, what information about the plasmids needs to be provided simultaneously?

A: The customer needs to provide the following information: the plasmid vector, the target sequence, and the insertion site. It would be best if the complete vector sequence containing the target gene could be provided. If you are very clear about the plasmid information, testing may not be necessary. If you are not very clear, it is recommended to arrange sequencing first.

Q2: What are the commonly used tags in mammalian systems?

A: Commonly used tags include Fc, His, and Flag tags. Adding an Fc tag can enhance expression levels. His and Flag tags are small tags and can be used for affinity purification.

Q3: What are the differences between CHO cells and HEK293 cells?

A: CHO cells (Chinese Hamster Ovary cells) are one of the most widely used and successful host cells for expressing recombinant proteins. They are commonly employed to produce recombinant proteins and monoclonal antibodies. CHO cells have well-characterized genomic information and demonstrate reasonable safety when handling human pathogenic viruses. Additionally, one of the key advantages of the CHO cell expression system is the ease of obtaining transgenic cell lines. However, due to glycosylation patterns that are not entirely identical to those in humans, recombinant proteins produced by CHO cells may still exhibit some degree of immunogenicity.

HEK293 cells, also known as Human Embryonic Kidney 293 cells, are a cell line derived from human embryonic kidney cells. They are one of the commonly used mammalian cell systems for protein expression, offering advantages such as rapid growth rate, high cell density, high transfection efficiency, and post-translational modifications that more closely resemble those of human proteins. Due to their excellent transfection efficiency, HEK293 cells are primarily used for transient expression. A drawback, however, is their relatively weak adhesion during growth, making them prone to detachment during experiments, which can affect experimental results.

Q4: What is the difference between transient transfection and stable transfection?

A: The plasmids used for transient transfection differ from those used for stable transfection. Plasmids for transient transfection do not require an antibiotic resistance gene, whereas those for stable transfection must include one to facilitate subsequent clone selection. Additionally, the culture media and experimental reagents used differ between the two methods.Transient transfection is simpler than establishing a stable cell line. After plasmid construction, the target protein can be obtained through cell thawing, transfection, cell culture, and protein purification. In contrast, establishing a stable cell line requires first linearizing the constructed plasmid, introducing it into cultured mammalian cells, and using an appropriate transfection method to integrate the plasmid into the host genome. Subsequently, steps such as pool selection, monoclonal screening, and cell passaging are necessary to obtain a stably transfected cell line. Once established, a stable cell line can produce the target protein consistently over the long term.

Q5: What are the common cell transfection methods and their technical principles?

A: As shown in the following table

Q6: If the expression level in transient transfection is low, how can the experimental protocol be adjusted to improve it?

A: Generally, protein expression is primarily determined by the protein's own characteristics. Common optimization methods include codon optimization, altering signal peptides, modifying expression vectors, cloning strategies (such as Fc tagging), analyzing cell lysates to determine protein secretion and expression, changing host cell lines, optimizing transfection methods, refining cell culture protocols, screening buffers during purification (precipitation, solubility), and adding protease inhibitors (if protein degradation is observed).