Expisf expression system

Manufactured by Thermo Fisher Scientific

The ExpiSf Expression system is a baculovirus-based expression system designed for high-yield production of recombinant proteins in Spodoptera frugiperda (Sf) insect cells. It enables efficient and scalable transient protein expression.

Automatically generated - may contain errors

Lab products found in correlation

Dh10bac competent cells, by Thermo Fisher Scientific (1 mentions) Pfastbac1 vector, by Thermo Fisher Scientific (1 mentions) Jem 1400, by JEOL (1 mentions) Pmsf protease inhibitor, by Roche (1 mentions)

3 protocols using expisf expression system

HuSaV GII.1 VLP Production

Check if the same lab product or an alternative is used in the 5 most similar protocols

The nucleotide sequence encoding VP1 of HuSaV GII.1 (sample ID 18003) was sequenced and cloned into the pFastBac1 vector (Invitrogen, Waltham, MA, USA). Each construct transformed DH10Bac competent cells (Invitrogen) and recombinant bacmid was purified. Recombinant baculovirus production was obtained by transfection of recombinant bacmid into ExpiSf9 cells using the ExpiSf Expression system (Thermo Fisher Scientific) and collecting the supernatant on day 5–6 of infection. Inoculating ExpiSf9 cells with this viral stock, the culture supernatant was harvested to confirm expression. The supernatant was centrifuged at 12,000× g for 10 min, and after repeated centrifugation, the supernatant was passed through a 0.45 μm filter (Merk Millipore, Burlington, MA, USA). After centrifugation at 113,000× g for 4 h, the precipitate was suspended in TNE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA [pH 7.4]) and left at 4 °C overnight. The samples were then suspended in 42% CsCl-TNE buffer and subjected to cesium chloride equilibrium density centrifugation at 139,000× g for 20 h, after which each fraction was collected and dialyzed against TNE buffer. The final sample was negatively stained with 1% uranyl acetate solution, and the VLP structure was confirmed by transmission electron microscopy (100 kV, JEM-1400, JEOL, Tokyo, Japan).

Matsumoto N., Kurokawa S., Tamiya S., Nakamura Y., Sakon N., Okitsu S., Ushijima H., Yuki Y., Kiyono H, & Sato S. (2023). Replication of Human Sapovirus in Human-Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells. Viruses, 15(9), 1929.

+ Open protocol

+ Expand

Recombinant Protein Production in ExpiSf9 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant proteins were produced with the ExpiSf expression system (Thermo Scientific). ExpiSf9 cells were grown in 1-liter shake flasks with 250 ml cell culture volume and were diluted to a density of 5 × 10⁶ cells/ml in medium supplemented with ExpiSf enhancer. At 18 to 24 h after dilution, the cells were infected with recombinant baculovirus at an MOI of 4 to 5 TCID₅₀ units per cell. The flasks were incubated for 2 to 4 days until the viability was reduced to 70%. Next, phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Roche) was added at a final concentration of 170 μg/ml. Cells were removed by centrifugation, and the pH of the resulting supernatant was increased to pH 7.8 by adding 0.1 M NaOH. The clarified supernatant was immediately used for purification or was stored at −80°C after the addition of glycerol (final concentration, 20%).

van Oosten L., Altenburg J.J., Fougeroux C., Geertsema C., van den End F., Evers W.A., Westphal A.H., Lindhoud S., van den Berg W., Swarts D.C., Deurhof L., Suhrbier A., Le T.T., Torres Morales S., Myeni S.K., Kikkert M., Sander A.F., de Jongh W.A., Dagil R., Nielsen M.A., Salanti A., Søgaard M., Keijzer T.M., Weijers D., Eppink M.H., Wijffels R.H., van Oers M.M., Martens D.E, & Pijlman G.P. (2021). Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants. mBio, 12(5), e01813-21.

+ Open protocol

+ Expand

Recombinant Protein Expression in Sf9 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

ExpiSF™ expression system (ThermoFisher: A38841) guidelines from the manufacturer’s was followed. In short, a pFASTBacDual expression plasmid was used to express either HA-Edc4 alone(N-terminally tagged) or αS-V5/HA-Edc4. These plasmids were transposed to DH10Bac™ competent cells. Proper integration of pFASTBac plasmids were screened both by blue-white screening and PCR analysis. The bacmid was purified with a modified miniprep protocol, making sure that the large bacmid is not sheared during the isolation. The bacmids were kept at 4°C no more than a week before the infection to avoid degradation. ExpiSf9™cells were cultured at 27°C in disposable Erlenmeyer flasks with vented caps with gentle shaking. The cells were kept at >90% viability measured by cell counting with Trypan blue. The bacmids were transfected to ExpiSf9™cells and the cells were observed for decline of growth and volume expansion. When the cell viability decreased to 60% and the cell diameter enlarged 20% (roughly to 20 um), the media was collected(P0). P0 viruses were directly used for infection (500 ul for 50 ml culture) and expression. After 3 days of infection, the cell pellet was harvested and kept at −80°C. Protein extraction and pulldown were carried exactly as in HEK293 cells (see below) except that cell lysis of Sf9 cells were carried out by repeated freeze-thawing.

Hallacli E., Kayatekin C., Nazeen S., Wang X.H., Sheinkopf Z., Sathyakumar S., Sarkar S., Jiang X., Dong X., Di Maio R., Wang W., Keeney M.T., Felsky D., Sandoe J., Vahdatshoar A., Udeshi N.D., Mani D., Carr S.A., Lindquist S., De Jager P.L., Bartel D.P., Myers C.L., Greenamyre J.T., Feany M.B., Sunyaev S.R., Chung C.Y, & Khurana V. (2022). The Parkinson’s disease protein alpha-synuclein is a modulator of Processing-bodies and mRNA stability. Cell, 185(12), 2035-2056.e33.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!