When using pFastBac1 for the expression of target proteins, His tags or GST tags can be added during plasmid construction, depending on the needs of the protein purification method and the need for fusion expression of the target proteins to increase their expression amount. To prevent the additional added protein tags such as His and GST from affecting the biological function and structural study of the target protein, a protease cleavage site, such as the cleavage site of TEV or PreScission, can be added between the fusion protein and the tag. If the protein is mainly expressed in soluble form, centrifugation collects insect cells and sonication lyses the cells, but various protease inhibitors need to be added because of the relatively large number of protease species in insect cells. Centrifugation collects the supernatant after sonication, and the target protein is purified using a nickel column or GST column, for example. The target protein can also be further purified using ion exchange, molecular sieve chromatography, etc. The purified proteins can be preserved by adding an appropriate amount of glycerol or, if necessary, can be concentrated and preserved.

Detailed Steps for Protein Expression Purification:

I. Generation and Characterization of Baculovirus

Cultivate the insect cells until the density reached 5×10⁶~1×10⁷ live cells/ml with ≥90% activity. Dilute the cells into 2.5×10⁶ viable cells/ml dilution with culture medium and recover at 27℃, 127 r/min for 25 min. Dilute the ExpiFectamineTMSf transfection reagent with Opti- MEMTMI serum-free medium and add Bacmid DNA (pc1 ⁃mfp5 as well as gus), incubate at room temperature for 5 min, and then dropwise add to 100 ml cell dilution. Incubate at 27℃, 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 P0 generation virus.

Serial 10-fold virus dilutions (10^-2, 10^-3, 10^-4, 10^-5) were prepared using ExpiSfTMCD medium and the cells were diluted to 1.25×10⁶ viable cells/ml in the medium. 1 ml of virus dilution was added to a 24-well plate (1 well for each dilution), followed by 1 ml of ExpiSfTMCD medium to a negative control without virus. negative control. 800 μl of cell suspension was added to 8 wells of virus dilution and negative control, and incubated in a shaker at 27°C, 227 r/min, protected from light for 14-16 h. Cells were removed from each of the 24 deep-well plates and centrifuged at 300×g for 5 min, and the supernatant was discarded. The cell precipitate was resuspended in 100 μl of diluted gp64APC antibody, vortexed for 3~5 s, incubated at room temperature for 30 min, washed with PBS and resuspended in 1 ml of fetal bovine serum dilution, and identified by flow cytometry.

II. Protein Expression and Characterization

Cells were cultured until the cell density reached 5×10⁶~1×10⁷ viable cells/ml with ≥90% activity, diluted with medium to a final density of 5×10⁶ viable cells/ml, immediately added with ExpiSfTM Transfection Enhancer, and incubated at 127 r/min on a shaking table at 27°C for 18-24 h. When the cell density was restored to 5×10⁶~7×10⁶ viable cells/ml with ≥80% activity, baculoviruses with multiplicity of infection (MOI) of 30, 60, and 120 were used, respectively. When the activity was ≥80%, the cells were infected with baculoviruses with multiplicity of infection (MOI) of 30, 60, and 120, respectively, and incubated at 127 r/min at 27°C until the cell activity decreased to less than 30%, and then centrifuged at 300×g for 5 min to remove the supernatant. 200 μl of cell supernatant and cell lysate were added to 50 μl of 5× SDS buffer, boiled for 6 min, subjected to polyacrylamide gel electrophoresis, and electrotransferred to acetate membrane at 90 V for 90 min.5% skimmed milk powder was closed for 1 h. Polyacrylamide gel electrophoresis was performed to verify the expression of the target proteins, and the purity of the proteins could also be verified by SDS-PAGE after purification, combined with gray scale analysis.

KMD Bioscience has a complete insect expression and purification system. The insect cell protein expression system is suitable for recombinant expression of membrane proteins, and transmembrane proteins with 4 times or less can be almost effectively expressed and purified in insect cells. Purification of membrane proteins is a time-consuming and labor-intensive matter, KMD Bioscience can provide customized products of full-length membrane proteins with high purity. We can provide customers with a complete set of protein expression solutions, we are free to sequence the genes provided by customers; at the same time, according to the codon preference of Sf9, Sf21, Hi-5 and S2 insect cells, free codon optimization, to effectively improve the expression of recombinant proteins, to provide you with one-stop service.