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:

HinkÕs 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 90Õ 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:

GraceÕs (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 (GraceÕs), NO pen/strep.

Marina started with 500mL at 3.3x106 with 98% viability

Spin down 10Õ, 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: