When using pFastBac1 to express a target protein, a His tag or GST tag can be added during plasmid construction, depending on the required protein purification method or the need to fuse the target protein to a tag to increase its expression level. To prevent the added His and GST tags from affecting the biological function and structural analysis of the target protein, protease cleavage sites—such as TEV or PreScission—can be inserted between the fusion protein and the tag. If the protein is primarily expressed in a soluble form, the insect cells are centrifuged to collect the cells, which are then sonically lysed; however, due to the presence of various proteases within the insect cells, various protease inhibitors must be added. Centrifuge the supernatant after sonication and purify the target protein using a nickel column or GST column. Additionally, ion exchange or gel filtration chromatography can be used to further purify the target protein. The purified protein can be stored after adding an appropriate amount of glycerol, or, if necessary, it can be appropriately concentrated before storage.

Detailed Steps for Protein Expression and Purification:

I. Baculovirus Preparation and Identification

    Culture insect cells until the density reaches 5×10^6–1×10^7 live cells/mL, with viability ≥90%. Dilute the cells with culture medium to a concentration of 2.5×10^6 live cells/mL, then recover at 27°C and 127 r/min for 25 minutes. Dilute the ExpiFectamine TMSf transfection reagent with Opti-MEM TMI serum-free medium, add Bacmid DNA (pc1-mfp5 and gus), incubate at room temperature for 5 minutes, and then add dropwise to 100 mL of the cell dilution solution. Incubate at 27°C and 127 r/min for 72–96 h until the cell viability drops to 60%–80%; centrifuge at 300×g for 5 min, and retain the supernatant as the P0-generation virus.

    Use ExpiSfTMCD medium to prepare a series of 10-fold viral dilutions (10⁻², 10⁻³, 10⁻⁴, 10⁻⁵), and dilute the cells in the medium to 1.25 × 10⁶ viable cells/mL. Add 1 mL of each viral dilution to a 24-well deep-well plate (one well per dilution), followed by 1 mL of ExpiSf™ MCD medium to the virus-free negative control well. Add 800 μL of cell suspension to the eight viral dilution wells and the negative control well, and incubate in the dark on a shaker at 27°C and 227 r/min for 14–16 h. Harvest the cells from the 24-well deep-well plate, centrifuge at 300 × g for 5 min, and discard the supernatant. Resuspend the cell pellet in 100 μl of diluted gp64APC antibody, vortex for 3–5 s, incubate at room temperature for 30 min, wash with PBS, resuspend in 1 ml of fetal bovine serum-diluted medium, and identify the cells using flow cytometry.

II. Protein Expression and Identification

    Cultivate cells until the cell density reaches 5×10^6–1×10^7 live cells/ml, with viability ≥90%. Dilute with culture medium to a final density of 5×10^6 live cells/ml, then immediately add ExpiSf™ transfection enhancer and incubate on a shaking incubator at 127 r/min and 27°C for 18–24 h. When the cell density recovers to 5×10^6–7×10^6 live cells/mL, and viability is ≥80%, infect the cells with baculovirus at multiplicity of infection (MOI) values of 30, 60, and 120, respectively. Incubate at 27°C and 127 r/min until cell viability drops below 30%, then centrifuge at 300×g for 5 min and collect the supernatant. Add 50 μl of 5× SDS buffer to 200 μl of cell supernatant and 200 μl of cell lysate, respectively; boil in water for 6 min; perform polyacrylamide gel electrophoresis; and transfer to an acetate fiber membrane at 90 V for 90 min. Block with 5% skim milk for 1 h, then perform polyacrylamide gel electrophoresis to verify the expression of the target protein. After purification, verification can also be performed via SDS-PAGE, and protein purity can be assessed through grayscale analysis.

    KMD Bioscience has a comprehensive insect expression and purification system. Our insect cell protein expression system is well-suited for the recombinant expression of membrane proteins; transmembrane proteins with four or fewer transmembrane domains can be effectively expressed and purified in insect cells. Since the purification of membrane proteins is a relatively time-consuming and labor-intensive process, KMD Bioscience can provide custom-made, high-purity full-length membrane protein products. We offer clients comprehensive protein expression solutions, including free sequence analysis and testing of customer-provided genes. Additionally, based on the codon preferences of Sf9, Sf21, Hi-5, and S2 insect cell lines, we provide free codon optimization to effectively increase recombinant protein yield, delivering a one-stop service.