Bacterial expression/His purification

Bacterial Expression w/His Purification

Expression of fusion proteins in bacteria (soluble proteins) �
By Jeremy Myers PhD
Updated: 10/12/06

Check Induction:

1. �� �Use bacteria transformed no more than 72hr prior to purification. (BL21DE3 RIL)
2. �� �Inoculate a 2ml culture from a single colony and grow with shaking 250rpm for 8hr or overnight at 37˚C - This is the �Starter Culture�.
3. �� �Use 50ul of the starter culture to inoculate a 1ml culture to test induction.
4. �� �Incubate 1.5hrs at 37˚C with shaking 250rpm. After which remove 100ul for the �uninduced sample� and store on ice.
5. �� � Induce the remaining culture by adding 1ul of 1M IPTG (1mM final conc.) and incubate 1.5 hr. After which remove 50ul for the �induced sample� and pellet uninduced and induced samples. �
6. �� �Remove supernatant, add 75ul 2XSB, and boil for 5min.� �
7. �� �After boiling centrifuge sample 10,000 rpm for 1min. �
8. �� �Load 12ul

Large Scale Induction:
Inoculate 500 ml of culture (LB pH 7.4) + 500ul Kan 500ul 25mg/ml Chloramphenicol with 2ml of and starter culture that was shown to be inducible.� Grow cultures until cultures are between 0.7-0.9 (check culture OD periodically to make sure not to over grow) usually take 3-4hrs. �

When bacterial culture reaches proper OD, Add 5ml of ethanol (1-5%, I use 2%) to maintain stress and chaperonin response.

After adding ethanol reduce culture temperature to 18˚C by cooling the bacteria for 7-12 minutes on ice.

Induce with 625ul of 0.2M IPTG (final � 0.25mM) for 500ml.� For 1ml of 0.2M IPTG use 43 mg in 1ml of ddH20� (64.5mg in 1.5ml)

Grow at 17.5˚C overnight (8-14hrs, I use 12%) - for increased solubility cultures must be aerated well � which means that agitation should be done at more than 215 rpms.

Spin cells down 5000xg 15 min at 4˚C.�� Store culture at -80˚C for at least 2hrs

Resuspend pellet in Sodium Phosphate buffer + EGTA for His purification each containing protease inhibitors PMSF, and phosphatase inhibitors NaF (imidazole is also an efficient phosphatase inhibitor).� Use 1/10 culture volume of buffer (for 500ml culture use 40-50ml).� Larger volume can be used and may improve purification

Next sonicate 4 times at settings 30% output 90% duty for 30 seconds each time. �

Cells should be lysed at this point and lysate should appear light-yellow and opaque. �

Add Sodium Phosphate Buffer � EGTA to 200ml and incubate 15 minutes on ice.

To determine solubility remove 20ul of lysate (prior to the following centrifugation step).� Centrifuge lysate 10000 rpm for 20min at �C. Remove supernatant and add 40ul of 2XSB. Resuspend the pellet (this will be very small) in 60ul of 2XSB.� Load 10ul of each.

Clear lysate by centrifugation at 14,000 x g (13,500 rpm) at 4 degrees for 20-30 min or 3000 x g 4˚C for 30min. �

HIS-SELECT PURFICATION BUFFER:
50mM Sodium Phosphate (monobasic)
400mM NaCl
2.5-5% Glycerol (optional � I use 5% flow rate may be faster with 2.5%)
Igepal CA-630 to a final concentration of 0.2% (use 1:100 of a 20% stock)
0.25mM EGTA � Add fresh only in lysis buffer
10mM Imidazole � Add fresh
0.5mM PMSF � Add fresh
1mM β-Mercaptoethanol or 0.2mM DTTafter lysozyme treatment

Phosphatase inhibitors can be added, however imidazole is an efficient phosphatase inhibitor
Adjust pH to 8.0 (this is necessary for His-Selection purification efficiency) and filter sterilize

DIALYSIS BUFFER (similar to kinase buffer):
10mM HEPES pH 7.5
50mM NACl
5uM B-glycerophosphate � Add fresh
0.25 mM DTT � Add fresh
0.05 mM PMSF

Make sure that you follow these steps:

IPTG is added to a logarithmically growing culture (OD600 between 0.5-0.9 with original OD600 less than 0.05).� Although this protocol calls for inducing at a specific OD this needs to be determined empirically. �

If the culture over grows before induction do not dilute. Start over. It is best to start the culture from a single colony in the morning, check induction, and add inducing for purfication before going home.

Cold Shock prior to adding the IPTG.

Make IPTG fresh

AERATION IS ESSENTIAL the volume of the flask should be at least 4 times the volume of the culture. 500 ml cultures should be in 2 liter flasks and it is important that you grow at least 500 ml for proteins with solubility issues.� Shaking at higher speeds (~215rpms) also helps.� Be sure that aeration is occurring shaking to fast can limit aeration.

The freezing step prior to lysis will increase your yield by helping the lysis step. If you do not want to freeze the pellet at -20 overnight place the bottle at -80 for two hours or so and allow the pellet to thaw on ice.

Less volume of lysis buffer is not better.� The concentration step will be with the sepharose.

You do not need to treat the proteins with DNAse I unless you are carrying assays that are sensitive to the presence of DNA in the prep.

Make the elution buffers fresh.� The elution step also helps concentrate the protein if done in small volume.

His-Select Purification (Sigma):

1. �� �Incubate cleared lysate with 400ul of His-Select resin for 20 min at 4˚C with rotation
2. �� �Add lysate to disposable Bio-Rad column and allow lysate flow through at a slow rate (one drop per second). This may take a few days and can be assisted by a pour man�s pump (syringe and tubing).
3. �� �Wash resin with (200x resin volume or ~100ml Sodium Phosphate buffer - EGTA).
4. �� �Elute with 54ml of Sodium Phosphate buffer plus 300mM Imidazole.� Collect 3 fractions

Protein should be exchanged into a new buffer to remove imidazole and to adjust salt levels. �

Addition of glycerol 50% is suggested for long term storage.

Tag can be cut off with TEV use 1/8 � 1/4 volume of 0.25ug/ul for one volume ~10 ug/ul protein. Incubate 4�C for 4 hrs.