Baculoviral-mediated protein expression in and purification from insect cells
10/12/04
SF9 cells from Marina in Harrison lab (6th floor). Came from Gibco (Bac to Bac system).
Cells double every 24-36 hours
You want to keep 0.7-2x106 per mL
Can also plate cells on 15cm plates at 1x106/mL, 20mL/15cm plate
Grow at 27C
Count cells, mix 100uL with 100uL trypan blue. Number of cells on 25 squares is
2x104/mL
Infect at 1.2x106/mL (so 0.7 on M pm is 1.2 on W am).
After infection keep 72 hours, then harvest
I have done 2.4L to get about 10-15mg of protein
In 12/03 the virus was titered at 2.4x108 (in 8/03). I use 3mL/600mL flask at 1.2x106,
which is an MOI of 1.
Can freeze down the cell pellet, store –80 until ready to purify.
SF21 medium:
Hink’s TNM-FH insect medium (JRH Biosciences 1-800-255-6032) 51942-1000M
Insect tested serum 10% Sigma F-3018
Pluronic 100X Sigma P5556
P/S/Q
Sf9 cells to prepare more virus:
Split with trypsin, wash with Ca/Mg free solution before trypsin
Plate at 0.7x106.
I have split confluent plate 1:3 on Friday, on Monday is about 80% confluent and ready
to infect
Infect 3/04 at 80% confluence
Do about a 1:1 MOI
3/04 - Original virus was 1x108, after 6 months at 4 degrees assume down by 25%, so I
assume 7.5x107. 11/04 – I assume after 8 months that virus is down even more so use
3mL virus with 1mL growth medium.
Mix 1mL virus with 3mL growth medium. Remove medium from 4 plates (11/04 I do 3
plates). Drip virus over plates (4mL for 15cm).
Rotate 90’ RT
Add 26mL growth medium (to 30 total per plate)
Put back for 48-72 hours
Cells should round up when they are ready to harvest.
I did at 72 hours.
In 4/04 we assume virus after harvest is 108, and test.
SF9 medium:
Grace’s (supplemented) Invitrogen 11605-094
Insect tested serum 10% Sigma F-3018
P/S/Q
Freezing SF9 cells:
Freezing medium: 7.5% DMSO, 10% FBS in base medium (Grace’s), NO pen/strep.
Marina started with 500mL at 3.3x106 with 98% viability
Spin down 10’, 4 degrees
Resuspend 5mL pellet in 18mL freezing medium
Put in cryovials and isopropanol cryofreezing containers. Leave greater than 24 hours at –
80 then to liquid N2.
Vector information:
pAcGHLT-A (Pharmingen)
Cloned full length mouse CaRF into EcoR1-Pst1
N-terminal GST and 6X His tag, linked to CaRF by PKA site and Thrombin cleavage site
We did not cleave off GST before rabbit injection.
Purification protocol: