1. E. coli protein purification*大見出し
I have several cDNA vectors for E.coli purification.
1. pQE vector (His tag)
2. pRSET vector (His tag)
3. pGEX vector (GST fusion protein)
What I need to prepare:
2. complete, EDTA free (Roche)
4. 10 mM, 20 mM, 50 mM, 200 mM and 1 M Imidazole PBS buffer
or 20 mM glutathione.
About His tag,
what is the column name?
About cell culturing:
0. Shake E.coli of interest overnight and make saturation.
1. put 2.5 ml E.coli in 250 ml LB (100 times dilution)
and 3-4 hour shaking @37C. It's OD value should be 0.6-0.8.
2. Add 1mM IPTG and shake 4 hour.
3. collect E.coli
4. Break E.coli with sonicator.
5. Spindown debris and pich up supernetant.
6. Add Ni++ beads or Glutathione beads and rotate 2 hour @4C.
2. Insect cell protein purification
Fisrt, this method is gonna be introduced in the case we cannot express the proteins of interest with E. coli.
Actually I struggled out to find out the way to express and purify the fusion protein of EYFP and C1ABgamma with E.coli. This protein seems to express with proper molecular weight when I check with SDS-page, but no fluorescence.
So that is why I used insect cell system.
This simple protocol is based on Bac-to-Bac Baculovirus expression system from invitrogen and from some tricks Karam told me.
First thing we need to do is that cDNA of interest is gonna be cloned in the FastBac vector, whose promotor is the Autographa californica multiple nuclear polyhedrosis virus promotor(AcMNPV:蛾核多角体病ウイルスのポリヘドリンプロモーター).
This expression cassette is flanked by the left and right arms of Tn7.
Tn7 is transposon, which moves freely and insert some place at random.
Tn7 in this system workd as something by which the gene of interest is transfered from FastBac vector to baculovirus shuttle vector (bacmid), which is in DH10Bac cells. DH10Bac cells include baculovirus shuttle vector and a helper plasmid.
1. prepare insert-containing FastBac vector.
2. transformation of this vector into bacmid in DH10alpha.
3. check whethere insert inserts into bacmid correctly by PCR.
4. transfection of bacmid into insect cells.
5. prepare P1 virus from supernetant of insect cells.
6. prepare P2 virus from supernetant of insect cells.
7. protein purification from insect cells.
Yoshi, this is the protocol.
Expression test (24-well)
The expression test is for the check of which P1 stock has maximum ability to express your protein.
1. Cells are growing in SF-900 II SFM with 5% FBS in suspension culture (log phase, ~1.0 x 106 cells/ml), with greater than 95% viability.
2. Seed 6 x 105 cells per well in a 24-well plate. Let cells attach for 30 minutes.
3. Remove the media and rinse the cells with fresh growth media (SF-900 II SFM with 5% FBS). Replace with 500 ul of fresh media.
4. Add 200 ul of P1 stock and incubate cells in the incubator.
5. Harvest cells at 48 hrs post-infection. Remove the media and rinse the cells with serum-free medium.
6. Lyse the cells with 100 ul of 1x SDS-PAGE buffer. Analyze protein expression.
Amplifying Viral Stock (P2 stock, 6-well plate)
1. Cells are growing in SF-900 II SFM with 5% FBS in suspension culture (log phase, 1.0~2.0 x 106 cells/ml), with greater than 95% viability.
2. Prepare 50 ml of 0.5 x 106 cells/ml suspension SF9 cells, and incubate it at 27oC for 1 day.
3. Add 500 l of P1 viral stock, and incubate it at 27oC for 4 to 5 days.
4. Collect the sup, and store it as P2 viral stock at 4oC. Titer is generally 1 x 107 ~ 1 x 108 pfu/ml.
Expression test 2
1. Prepare three 50 ml culture SF9 cells, and incubate for 1 day. The concentrations of these cells are 0.8 x 106 cells/ml.
2. Add variable amounts of P2 stocks to each bottle. Usually, we add 0.5, 1.0 and 2.0 ml of P2 stocks in each bottle.
3. You can harvest cells at 48 hours after infection. Use 1ml of cells for expression check (WB). Collect remaining cells, and rapidly freeze them with liquid N2. Store it at -80 oC. If expression level of your protein is good, you could use it for purification.
1. A day before infection, cell concentration is 0.8 x 106 cells/ml (100~500 ml).
2. Add appropriate amounts of P2 stock to cells, and incubate it for 3 days at 27oC.
3. Harvest cells.
1.At first, check insect cells that these have fluorescence.
2.Spine down these cells with 3000g for 5 min.
3.Suspend these cells in 10 ml 10 mM imidazole PBS(-) containing inhibitor cocktail.
4.Break cells with sonication, amplitude 3, 30 sec, 3 times.
5.Spine down these debris wit 15000g for 10 min.
6.Add Mi++-NTA beads into 10 ml solution.
7.incubation and rotate for 2 hours.
8.Put these solution on the coloum(poly-prep chromatography columns:731-1550 columns, 0.8*4cm Bio-lad)
9.Wash these gel with 1 ml 20 mM imidazole buffer
10.Wash these gel with 1 ml 50 mM imidazole buffer
11.Elute solution with 200mM imidazole 1ml.
How long does it takes for getting protein of interest?
In my impression, at least one month for beginner.
How do we keep stock virus solution?