蛋白纯化操作

His-tag融合蛋白的纯化

The following protocols is for His Bind resin

His Bind Purification

1) Express and harvest cells. Resuspend cells in Binding Buffer. Lyse cells.

2) Prepare resin (~20- 25 ml?) in column - wet column and frit, transfer resin slurry to column. Wash with water (3 volume), Charge Buffer (5 volumn), Binding Buffer (3 volume).

3) Load column with cell lysate, at a rate of ~10 column volumn per hour.

4) Wash column (10 X volumn) with Binding Buffer

5) Wash column (6 X volumn) with Wash Buffer

6) Elute protein (6 X volume) with Elute Buffer (strip buffer can also be used to elute). Alternatively elute first with lower concentration of imidazole at 0.25 M.

7) To store column, wash with Strip Buffer (3 X volumn) and stored in Strip Buffer at 4 °C.

1X Buffer

Purification in Denaturing Condition (protein in inclusion body)

1) Same as before.

2) Centrifuge to collect inclusion body. Decant supernatant, resuspend in Binding Buffer (sonicate if necessary), centrifuged. Repeat until all trapped proteins are released.

3) Decant supernatant, resuspend in Binding Buffer + 6 M guanidine or Urea. Incubate on ice for 1 hour to dissolve.

4) Centrifuged at 39,000 g for 20 min. Filter supernatant (0.45 micron membrane).

5) Load onto column. Purification same as before, except that all buffer contain denaturant, and lower imidazole concentration in Wash Buffer (20 mM) and Elute Buffer (~300mM) (dilute Wash and Elute Buffer with Binding Buffer). Alternatively, you can wash and elute the protein without imidazole at low pH (wash at pH 6.5, elute at pH5.9 or pH4.5 if the protein failed to elute at the hight pH)

Regeneration of Column

- when flow rate is slow and column resin doesn't turn blue-green when charged

1) Wash with 2 vol 6 M guanidine-HCl, then 3 vol water.

2) Wash with 1 vol 2% SDS.

3) Wash with 1 vol each of 25%, 50% and 75% ethanol, then 5 vol of 100% ethanol, followed by 1 vol each of 75%, 50% and 25% ethanol.

4) Wash with 1 vol water, then 5 volume 100 mM EDTA (pH 8.0).

5) Wash with 3 vol water, then 3 vol 20% ethanol.

6) Store at 4 °C.

Note:

1) Do not use ?ME, DTT or EDTA in buffer.

2) Higher amount of imidazole may be used in the wash buffer (100 mM) but some protein may be eluted.

3) You can also elute the protein with salt concentration gradient or using lower pH.

4) It's sometimes useful to introduce a couple of glycines between your protein and the His-tag which allow the his-tag to be fully exposed for effective binding to resin (if your vector doesn't have a short linker sequence between the HIs-tag and your protein that is).

His-tag融合蛋白纯化中Ni柱的使用Materials and reagents

Extraction buffer

Preparation of the magnetic beads

Procedure

GST融合蛋白纯化方法

Abstract: Many people have vented out frustration over insoluble GST-fused proteins. This is a protocol for enzymatically active soluble GST-fused proteins. All GST-fused proteins are rendered soluble with this technique though enzyme activitiy can range from 30-90%.

Materials and Reagents

1.STE Buffer

10 mM Tris-HCl, pH 8.0

1 mM EDTA

150 mM NaCl

2.Lysozyme solution

10 mg/ml in water (make fresh)

3.PBS

4.Elution Buffer

50 mM Tris.Cl, pH 9.0

20 mM GSH

5.10% Sarkosyl in STE Buffer

6.10% Triton X-100 in STE Buffer

7. 1 M DTT

8.100 mM IPTG

Procedure

Day 1

1.Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin.

Day 2

1.Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicillin.

2.Grow at 37o C to an A600 of 0.6 to 0.8.

3.Induce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr at 37o C or grow overnight at room

temperature.

Lower IPTG concentrations and lower growing temperatures tend to produce greater solubility at the expense of yield.

4.Pellet cells by centrifuging at 3000 g, 4o C for 10 min. Decant media and resuspend cells

in 30 ml ice-cold PBS to wash. Transfer to a 40-ml Oak Ridge tube and centrifuge at 3000 g, 4o C for 10 min. Decant PBS.

5.This is a convenient point to stop and to store pellets at -80o C. Else continue to lyse

cells.

6.Thaw pellet on ice if cells are frozen else proceed to the next step.

7.Resuspend pellet in 10 ml of ice cold STE Buffer.

8.Add 100 ml of freshly prepared lyozyme solution, incubate on ice for 15 min. Just

before sonication, add 100 ml of 1 M DTT and 1.4 ml of 10% Sarkosyl. Mix thoroughly by inversion and sonicate for a total time of 1 min.

9.Centrifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Transfer

supernatant to a 50-ml conical tube and discard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20 ml. The effective concentration of Sarkosyl and Triton X-100 will be 0.7% and 2% respectively. Incubate at room temperature for

30 min.

10.Pour the lysate to 1 ml bed of prepared Glutathione Sepharose in PBS. Incubate at

room temperature for 30 min to 1 hr with agitation.

To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml

tube. Fill to 50 ml with PBS, invert tube a few times. Centrifuge to 2000 rpm

on a swing bucket centrifuge then switch off. Carefully suck off PBS and

resuspend beads with 1 ml of PBS.

11.Wash the beads with 3 X 50 ml of PBS. Finally resuspend in 5 ml of PBS. Pour to a

dispo-column. Wash the 50-ml conical tube with an additional 5 ml of PBS. Pool with

the first 5 ml in the dispo-column.

To wash, use the same centrifugation technique for preparing the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick

to pipette tips.

12.If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions

with SDS PAGE.

Reference

Frangioni and Neel.Anal. Biochem. 210, 179-187 (1993)

酵母中GST融合蛋白纯化方法

? 5 ml overnight culture of your favorite yeast in your favorite medium.

?Inoculate 50 ml and grow 30o C shaking O/N until OD600 = 0.8 to 1.2. For SCD cultures use 1/500 and 1/1500 dilutions.

?Optional: add alpha-factor to 2.5 μM. Continue shaking at 30o C for 60 min.

?Add 1 M NaN 3 to 10 mM (final concentration) and move cultures to ice. Everything must remain cold from here on out.

?Spin cells 3 K, 10 min at 4o C.

?Discard supe and resuspend cells with 0.5 ml 10 mM NaN3. Transfer to 1.5 ml microfuge tube (not autoclaved).

?Spin 3 K, 10 min at 4o C. Alternatively spin 8 K, 1 min at room temperature.

?Discard supe. Optional: freeze pellet at -80o C.

?Resuspend in 1 ml of cold 10 mM NaN3.

?Measure OD600. Adjust volumes so that there are an equal number of cells in each sample. A total OD600 of 30 per sample is best.

?Wash with 1 ml of Lysis Buffer.

?Spin 3 K 10 min at 4o C and discard supe.

?Resuspend in 400 ul Lysis Buffer.

?Add a scoop of glass beads to a 0.5 ml PCR tube. Transfer cell lysate to the PCR tube

?Vortex 1 min, 4X. Keep samples cold between vortexing.

?Poke a hole in the bottom of the tube and spin cell lysate in a new microfuge tube 1.5 K or 500 X g, 10 min., 4o C.

?Transfer liquid from bottom tube into a new microfuge tube.

?Spin again 1.5 K, 10 min, 4o C and again transfer liquid into a new microfuge tube.

?Add Triton X-100 to 1.5 % and rock for 60 min at 4o C.

?Spin (3 K, 10 min, 4o C) and transfer the supe to a new microfuge tube.

?Remove 30 ul of liquid and add 30 ul 2X SDS PAGE Sample buffer. This will reflect protein content before Glutathione purification.

?To the remaining liquid, add 100 ul 40 % slurry of Glutathione beads and mix at 4o C for 2 h (overnight is usually fine). Glutathione beads should be prewashed 3 X with PBS and 1 X with lysis buffer before resuspending as a 40 % slurry in lysis buffer.

?Wash glutathione beads five times with PBS, 1 % Triton X-100, 300 mM NaCl at RT. Spin 2 K, 5 min, at room temperature. Rock sample for 5 min between washes. Change tubes after the first wash to reduce nonspecific binding to the tube itself.

?Resuspend in SDS-PAGE Sample buffer.

o Alternatively elute 3 times with 1-2 column volumes of 5-10 mM reduced glutathione, 50 mM Tris pH 8, and mix with 6X SDS-PAGE sample buffer

before stripping the beads with SDS-PAGE sample buffer.

?Heat to 100o C for 10 min. Then store at -20o C. Protein is ready to be run on SDS-PAGE Gel.

NOTES

Thanks to Paul DiBello and Jiyoung Cha for their refinements of this protocol.

* Indicates a correction from an eariler version of the protocol.

1. For lysis in the presense of GDP and GTP I use a final concentration of 10 uM GDP or 20 uM GTPgammaS and a final concentration of 3 mM MgCl2 in the lysis buffer.

2. You can substitute protease inhibitor cocktail (Sigma P8215) for individual protease inhibitors (AEBSF, leupeptin, pepstatin, benzamidine, aprotinin).

3. For lysis to determine phosphorylation, you may wish to add more phosphatase inhibitors (in addition to beta-glycerolphosphate and Na-o-vanadate) to the lysis buffer, or you may wish to omit phosphatase inhibitors altogether:

50mM Na-M-Vanadate 200ml 0.5mM

100mM

2 ml 10mM

Na-pyrophosphate

2mg/ml Phosvitin 10μl 1μg/ml