Workflow for Protein Expression in Mammalian Cells

2026-07-09

201

Expression systems used for recombinant protein production primarily include prokaryotic expression systems, yeast expression systems, insect cell expression systems, and mammalian cell expression systems. Compared to other expression systems, mammalian cells are capable of performing complex post-translational modifications, such as glycosylation and proper protein folding, resulting in target products that more closely resemble natural molecules; as such, they have become an important host for recombinant protein expression.

Generally speaking, the types of proteins suitable for mammalian expression include secreted proteins, antibodies, extracellular domains of transmembrane proteins, and extracellular domains of viral glycoproteins. The most common mammalian expression systems are Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells.

Workflow for Protein Expression in Mammalian Cells

Plasmid Extraction	a. Using the four-zone streaking method, streak a small amount of bacterial suspension onto Amp- or Kana-resistant LB plates; b. Incubate in a constant-temperature incubator; c. Pick a single colony and inoculate it into LB liquid medium containing Kana or Amp resistance; d. Incubate in a shaking incubator; e. Perform plasmid extraction using a plasmid extraction kit (refer to the kit instructions for details); f. Collect the plasmid DNA;
Cell Transfection	a. 24 hours before transfection, seed the cells using fresh complete medium; b. On the day of transfection, the cell density in the suspension culture flask should be 1×10^6 cells/ml, and the live cell rate as determined by trypan blue staining should be greater than 95%; c. Add an appropriate amount of complete medium and the plasmid to a sterile centrifuge tube, and mix by vortexing; d. Add the transfection reagent to the plasmid mixture, vortex to mix, and incubate at room temperature; e. Add the incubated transfection complex dropwise to the suspension culture flask; f. Culture in a CO₂ incubator at the appropriate temperature, humidity, and CO₂ concentration; place the suspension culture flask on a shaker and maintain suspension culture at a specific rotation speed; g. 4–5 days after transfection, centrifuge to collect the expressing cells and supernatant for protein purification;
Protein Purification	a. Load the column; b. Equilibrate the column: Use the equilibration buffer to equilibrate the column at a specific flow rate until the baseline of the purification system stabilizes; c. Sample loading: Once the baseline has stabilized, pass the prepared purification sample through the column at a specific flow rate. After the sample has been fully eluted, use the equilibration buffer to elute the unbound components until the baseline of the purification system stabilizes; d. Elution: Collect all elution peaks separately into sterile centrifuge tubes; e. Dialysis: Dialyze overnight in standard buffer;
Forensic Analysis	SDS-PAGE、WB