EarlyBac manual

References

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

Vectors (click on items for sequence info/maps)

pFp10

pUCDMp10

pSPLp10

  pFp10* pUCDMp10 pSPLp10
One protein*    
Two proteins**  
Two proteins**  
Three proteins

*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

*Low copy

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 do two vectors at a time (three may be done with low efficiency).

First round

pFp10 + pUCDMp10 pFp10/pUCDMp10

pFp10 + pSPLp10 = pFp10/pSPLp10

Second round

pFp10/pUCDMp10pSPLp10 = 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.

-Use Cellfectin II (Thermofisher) as a transfection reagent.

 

Bacmids

-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)

 

Transfection/Virus amplification

-Sf9 cells (e.g. 6 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 Sf-900-III since titers are higher.

-Large scale expression can be done in other media such as ESF921.

 

Mid-large scale expression

-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).

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).

 

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