Effective production of oligomeric membrane proteins by EarlyBac-insect cell system
H Furukawa, N Simorowski, K Michalski
Methods in Enzymology in press, 2021
Structural basis of functional transitions in mammalian NMDA receptors
TH Chou, N Tajima, A Romero-Hernandez, H Furukawa
Cell 182 (2), 357-371. e13, 2020
Plasmids are deposited to Addgene
Vectors (click on items for sequence info/maps)
*Always subclone your first gene into pFp10. If you are expressing one protein, subclone into pFp10
**Subclone your 2nd gene into pUCDMp10 or pSPLp10
Amplification of plasmid vectors
pFp10 – use DH5alpha – Ampicillin resistance
pUCDMp10* – use PIR1 from Thermofisher (cat#: C101010) – Chloramphenicol resistance
pSPLp10* – use PIR1 from Thermofisher (cat#: C101010) – Spectinomycin resistance
The above vectors were made in the backbones of pFL, pUCDM, and pSPL multibac vectors ( Fitzgerald et al Nat Methods. 2006 Dec;3(12):1021-32. doi: 10.1038/nmeth983.)
Cre-lox recombination (for multiple protein expression)
-We typically recombine two vectors simultaneously (three may be done with low efficiency).
pFp10 + pUCDMp10 = pFp10/pUCDMp10
pFp10 + pSPLp10 = pFp10/pSPLp10
pFp10/pUCDMp10 + pSPLp10 = pFp10/pUCDMp10/pSPLp10
pFp10/pSPLp10 + pUCDMp10 = pFp10/pSPLp10/pUCDMp10
-We make our own Cre-recombinase, but it can also be purchased from NEB (https://www.neb.com/products/m0298-cre-recombinase#Product%20Information)
Quick expression test by transient transfection
-Quick expression experiments (e.g., FSEC) can be done by transfecting the vectors into insect cells before making virus.
-We transfect into HighFive cells for FSEC since the transfection efficiency is higher than Sf9 cells. Culture HighFive cells in ESF921.
-Use Cellfectin II (Thermofisher) as a transfection reagent.
-We happen to use DH10EmBacY (Geneva-Biotech, https://geneva-biotech.com/product_category/insect-cell-expression/multibac/). This bacmid expresses YFP, which serves as an infection marker. The viral genes v-cath and chiA are also disrupted.
-DH10Bac (Thermofisher, https://www.thermofisher.com/order/catalog/product/10361012?us&en#/10361012?us&en) can also be used.
-Transposition can be done using the methods described in Thermofisher or Geneva technology (https://geneva-biotech.com/wp-content/uploads/2019/12/MultiBac-Manual-v8.4.pdf)
-Sf9 cells (e.g., six-well plates containing 3X10^6 cells in 2 ml of media per well)
-We use TransIT-2020 or Fugene for transfection.
-Wait 4-5 days after transfection and collect supernatant (P1 virus).
-We like to do transfection and virus amplification in Hyclone CCM3 medium.
Mid-large scale expression
-We culture Sf9 cells in Hyclone CCM3 medium. We have not done an extensive media screening but see a higher expression level in CCM3 than in Sf900-III when using EarlyBac.
-For membrane protein expression, we grow Sf9 cells to 4X10^6 cells/ml (0.8-1 L in 2 L flask; 110 rpm at 27oC).
-For secretion expression, we grow HighFive cells to 1.5-2X10^6 cells/ml (0.8-1 L in 2 L flask; 110 rpm at 27oC). Use ESF921 medium for culturing HighFive cells.
By default, we collect cells 48-h post-infection. Remember, you are using an early promoter, not a late promoter; thus, the protein expression starts immediately after infection (~12 h).