Q1. Under what circumstances is it appropriate to use the mammalian system for antibody recombinant expression?

A1: The essence of antibodies is immunoglobulins. When expressing eukaryotic proteins, the corresponding expression system will be selected according to different eukaryotic protein expression requirements. The mammalian cell expression system can provide a cell environment similar to the natural cell environment, which is suitable for the expression of complex eukaryotic proteins. In addition, the mammalian protein expression system can synthesize proteins with similar structures and functions to human proteins. The recombinant expression of antibodies by the mammalian expression system can ensure correct post-translational modifications, such as glycosylation. The mammalian cell expression system can express a large number of proteins, which is suitable for large-scale recombinant antibody production. In addition, the proteins expressed by mammalian cells can be secreted outside the cells, making the recombinant protein expression and purification process simpler. Based on the powerful post-translational modification function of the mammalian cell expression system, mammalian protein expression is also closer to the structure of human proteins. Therefore, the mammalian expression system is often used for therapeutic antibody development.

Q2. How to analyze the protein sequence of a given antibody?

A2: First, find the protein information and its amino acid sequence according to the protein and species to be expressed by the customer. Then, transmembrane analysis, signal peptide analysis, and hydrophilicity analysis are performed. If the protein contains a signal peptide region, the signal peptide region needs to be removed during expression. When there is a transmembrane domain in the protein, an insect expression system is generally used, which can span up to 7 times. By observing the peaks of the image generated in the hydrophilicity analysis, it is possible to determine whether the protein as a whole is hydrophilic or hydrophobic. If it is a hydrophobic protein, a SUMO tag needs to be added to promote solubility. After obtaining the above relevant information, the subsequent expression system, enzyme cleavage site, tag, and vector selection can be carried out.

Q3. How to design recombinant antibody expression?

A3: The principle of recombinant protein expression is mainly to clone the target gene into an expression vector and transform it into a host cell, and then produce the target protein through transcription and translation. When producing antibodies, commonly used expression vectors include plasmids and viral vectors. Promoters and transcription termination signals can be inserted into the expression vector to ensure that the transcription and translation of the gene are carried out efficiently. The antibody gene is inserted into the appropriate position of the vector, and then the constructed vector is transfected into the host cell. The marker can be used to screen the stable expression cell line of the antibody for large-scale recombinant antibody preparation. Commonly used mammalian cells for antibody expression include CHO cells and HEK293 cells. Adding His tags, FLAG tags, etc. is conducive to affinity purification of antibodies.

Q4. What are the commonly used mammalian cell transfection methods?

A4: When transfecting mammalian cells, transient transfection or stable transfection is mainly used. Transient transfection is simple to operate and can express the target gene in a short time. It is mostly used for small-scale synthesis of proteins in a short period of time. Stable transfection can be used for long-term experimental research and development of mammalian cell antibodies. Cell transfection methods mainly include physical transfection (electroporation, microinjection and gene gun, etc.), chemical transfection (liposomes and their substitutes, calcium phosphate, etc.), and biological transfection (various viruses, including adenovirus, lentivirus, retrovirus, adeno-associated virus, etc.).

Q5. What should be paid attention to when culturing mammalian cells?

A5: During the cell culture process, it is important to pay attention to aseptic operation to prevent cell contamination. When reviving cells, the mouth of the cryopreservation tube should not touch the water surface to reduce the possibility of contamination by impurities in the water. When performing cell passaging, the time of trypsin digestion should be adjusted according to the cell type and cell state. When resuming cells and freezing and thawing, pay attention to "slow freezing and fast thawing" to reduce temperature damage to cells. When culturing cells, the state of the cells should be checked every day, and the cells can be passaged when they grow well and have a moderate density.

Q6. What are the common types of cell contamination? How to prevent it?

A6: Depending on the source of contamination, cell contamination is mainly divided into physical contamination, chemical contamination, and microbial contamination. Overcooling or overheating can cause changes in cell state and even cause cell death. Cells exposed to radiation and radiation will destroy the DNA structure of cells, leading to cell death or genetic mutations. Impure components or concentration problems such as culture medium, water, and reagents may affect cell growth and even produce toxic effects. Unqualified reagents used when treating cells will also affect cell growth. Microbial contamination is a relatively common and serious contamination in the cell culture process. According to the type of microorganism, it can be divided into bacterial contamination, fungal contamination, mycoplasma contamination, and viral contamination. Once cells are contaminated, they will affect the state of cells, prolong the cell culture cycle, and affect the experimental results. Therefore, the cell culture environment should be kept clean and tidy, and disinfected regularly. At the same time, during the operation, relevant regulations should be strictly followed to ensure aseptic operation.

Tek Biotech has established a complete recombinant antibody mammalian expression system, including but not limited to FreeStyle 293-F cell line, Expi 293-F cell line, Expi-CHO-K1 and Expi CHO-S cell lines, and cooperates with the mammalian expression vector designed by Tek Biotech (containing the full-length CMV promoter and the optimized secretion signal peptide sequence), which can provide customers with higher expression levels of recombinant antibody mammalian cell expression and recombinant antibody production services. In addition, Tek Biotech has a mature vector-cell-transfection efficient expression system, which can shorten the experimental cycle and save time for customers.