Compared with other systems, the mammalian cell expression system has the greatest advantage of guiding the correct folding of proteins, providing multiple post-translational processing functions such as complex N-type glycosylation and accurate O-type glycosylation. Therefore, the expressed product is closest to the natural higher biological protein in terms of molecular structure, physical and chemical properties and biological functions. Despite its complex operation, low yield and high culture cost, it is still the preferred method to obtain highly active biological proteins. In some cases, the activity of its expression products is far superior to that of prokaryotic expression systems and eukaryotic expression systems such as yeast and insect cells. The most commonly used host cells in conventional experiments are HEK 293 cells and CHO cells. The former is generally used for transient expression of recombinant proteins to obtain a small amount of bioactive recombinant proteins in a short period of time. The latter is generally used for stable expression of recombinant proteins. CHO cells play an important role in industrial production and medical treatment due to their advantages such as stable genetic traits, fast growth rate, tolerance to certain shear force and osmotic pressure, and glycation modification similar to human beings.

Our company provides HEK293 cell transient protein expression service and CHO cell stable strain construction service, in which the HEK293 cell transient expression process is as follows:

1. Plan design - analyze target gene sequence, and make experimental plan according to customer requirements and existing literature reports. The company provides free of charge for customers: protein signal peptide analysis, protein hydrophobicity analysis, protein transmembrane analysis, glycation site prediction, GPI function anchoring, etc.

2. Construction of recombinant plasmids within 1-2 weeks. Appropriate vectors and appropriate labels (eg:His,GST, FC, SUMO) were selected to construct recombinant plasmids, supporting site-directed mutagenesis, vector modification, seamless cloning and other services.

3 cells were transfected into suspension HEK293 cells after 1 week of recovery and subculture. After the recombinant plasmid was extracted, it was transfected into suspension HEK293 cells using chemical transfection reagent.

4. Separation and purification of recombinant proteins After 1 week, appropriate chromatographic methods were used to isolate and purify the recombinant proteins, and the ultrafiltration solution was changed to the buffer solution specified by the customer (PBS or Tri-HCl was selected as the final buffer solution if there was no special requirement). The more commonly used chromatography methods are: affinity chromatography (Ni, GST), anion, cation exchange chromatography (Q, SP), molecular exclusion chromatography, etc.

5 QC analysis of the recombinant protein for 1 week SDS-PAGE electrophoresis was used to detect the purity of the recombinant protein, the concentration of the recombinant protein was quantified by BCA, and the immunogenicity of the recombinant protein was tested by Western-blot.

6. Recombinant protein was reprocessed for 1 week (optional, extra charge is required). Recombinant protein was filtered and sterilized, endotoxin removed, label cutting, etc.;

Advantage: Our company has a mature and powerful HEK 293 transient expression system. Through accurate protein structure prediction and combined with literature reports, we have expressed a variety of membrane proteins with complex structure (such as CD3, CD20, HER2, EGFR, CD19, PD-L1, VEGF165, CD105, EpCAM, FAP, GP73, etc.). Combined with our excellent downstream purification process, we have the ability to express and purify recombinant proteins (such as human calcium receptor protein: CASR, with an expression level of about 200ug/L) per microgram per liter.

The construction process of CHO stable cell line is as follows:

1. Plan design - analyze target gene sequence, and make experimental plan according to customer requirements and existing literature reports. The company provides free of charge for customers: protein signal peptide analysis, protein hydrophobicity analysis, protein transmembrane analysis, glycation site prediction, GPI function anchoring, etc.

2. Construction of recombinant plasmid within 1-2 weeks; select the right vector and construct the recombinant plasmid with the right label; support site-directed mutagenesis, vector modification, seamless cloning and other services;

3. cells were transfected into CHO cells in suspension culture after 4-5 weeks of resuscitation and passage. After the recombinant plasmid was extracted, the cells were transfected into CHO cells in suspension by physical or chemical methods, and the first round of "Stable pool" was obtained by selecting medium.

4. Screenings of high-yielding cell lines 3-5 weeks after the first round of "Stable pool" was dispersed to 96-well plates to form monoclones, and then high-yielding cell clones were screened by appropriate methods (such as ELISA, SDS-PAGE, etc.);

5. Shaking flask test of high-yielding cell clones After 6-8 weeks, the high-yielding cell lines were further screened by shaking flask small-volume tracking method, and their passage stability was preliminarily studied. At this time, a subclone could be carried out if possible.

6. Separation and purification of recombinant protein 1-2 weeks (optional) The appropriate chromatographic method was used to isolate and purify the recombinant protein;

Advantages: Our company has a strong industrial CHO GS expression system (i.e., "Glutamine Synthase Screening System"), and the CHO antibody stable cell lines constructed by this screening system can express water up to 500 mg-1000 mg/L.