(整理)GST融合蛋白纯化方法.

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GST

融合蛋白纯化方法

Purification of GST Fused Proteins

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 , 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.

G

row at 37

o

C to an A

600

of 0.6 to 0.8.

3.

I

nduce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr at 37

o

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.

P

ellet cells by centrifuging at 3000

g

, 4

o

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

, 4

o

C for 10 min. Decant PBS.

5.

T

his is a convenient point to stop and to store pellets at -80

o

C. Else continue to lyse cells.

6.

T

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

7.

R

esuspend pellet in 10 ml of ice cold STE Buffer.

8.

A

dd 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.

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9.

C

entrifuge 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.

PAGE

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

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亲和层析实验技术方法

INTRODUCTION

This protocol describes a method for removing antibodies that react with bacterially encoded proteins by

passing

a

crude

preparation

of

immunoglobulins

through

a

column

containing

immobilized

bacterial

proteins.

MATERIALS

?

Reagents

E. coli strain used as host for preparation of expression library

Antibody preparation that is to be used for screening

This

protocol

works

best

when

using

an

IgG

fraction,

prepared

by

chromatography

of

the

antiserum

on

protein A-Sepharose.

?

Cell lysis buffer

0.1 M sodium borate (pH 8.0)

1 M NaCl

Sterilize the cell lysis buffer using a 0.45-?

m filter, and store at room temperature. Approximately 100 ml

of cell lysis buffer is required per 1 liter of bacterial culture.

?

Growth medium

One liter of growth medium appropriate for the E. coli strain of choice is required.

?

Lysozyme

Dissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately before

use. Make sure that the pH of the Tris solution is 8.0 before dissolving the protein. Lysozyme will not

work efficiently if the pH of the solution is less than 8.0.

Use a molecular biology grade of lysozyme. Add solid lysozyme to assist lysis of bacterial cells.

?

NaOH (1 N)

The

preparation

of

10

N

NaOH

involves

a

highly

exothermic

reaction,

which

can

cause

breakage

of

glass containers. Prepare this solution with extreme care in plastic beakers. To 800 ml of H

2

O, slowly

add 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. When

the pellets have dissolved completely, adjust the volume to 1 liter with H

2

O. Store the solution in a plastic

container at room temperature. Sterilization is not necessary.

?

Pancreatic DNase I

o

1 mg/ml Pancreatic DNase I

o

50 mM NaCl

o

10 mM Tris-Cl (pH 7.5)

o

1 mM MgCl

2

Dissolve 2 mg of crude pancreatic DNase I (Sigma or equivalent) in 1 ml of 50 mM NaCl, Tris-Cl (pH 7.5),

1

mM MgCl

2

. When the DNase

I is dissolved, add 1

ml of glycerol to

the solution and

mix by

gently

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inverting the closed tube several times. Take care to avoid creating bubbles and foam. Store the solution

in aliquots of -20°

°

C. Add solid DNase I to the bacterial cell lysate to digest chromosomal DNA.

Tris-buffered Saline (TBS)

?

Dissolve 8 g of NaCl, 0.2 g of KCl, and 3 g of Tris base in 800 ml of distilled H

2

O. Add 0.015 g of

phenol red and adjust the

pH to 7.4 with HCl. Add distilled H

2

O to

1

liter. Dispense

the solution

into

aliquots and sterilize them by autoclaving for 20 minutes at 15 psi (1.05 kg/cm

2

) on liquid cycle. Store the

buffer at room temperature.

?

?

TBS containing 0.2% (w/v) sodium azide

Triton X-100

METHOD

Grow a 1-liter culture of the appropriate strain of

E. coli

(e.g., Y1090hsdR, XL1-Blue, or DH1) to

stationary phase.

?

Recover the bacteria by centrifugation at 4000g (5000 rpm in a Sorvall GSA rotor) for 20 minutes at

4°

C.

?

Pour off the medium, and stand the centrifuge tubes in an inverted position to allow the last traces of

medium to drain away.

?

?

?

?

Resuspend the pellet in 100 ml of Cell lysis buffer.

Add 200 mg of lysozyme, and incubate the bacterial suspension for 20 minutes at room temperature.

Add 1 mg of pancreatic DNase I and 200 ?

l of Triton X-100.

Incubate

the bacterial suspension

for 1

hour at 4°

C, or until the

turbidity clears and

the

viscosity

decreases.

?

Centrifuge the bacterial lysate at 8000g (8200 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4°

C.

Carefully decant the supernatant into a fresh flask.

?

?

Adjust the pH of the supernatant to 9.0 with 1 M NaOH.

Determine the concentration of protein in the lysate using the Lowry, Bradford, or other method of

measurement.

?

Chill

the

extract

to

0°

C,

and

then

bind

the

bacterial

proteins

to

cyanogen-bromide-activated

Sepharose 4B or to Affi-Gel 10 according to the manufacturer's instructions.

?

Before use, equilibrate the Sepharose 4B or Affi-Gel 10 resin containing conjugated E. coli proteins

in TBS containing 0.2% (w/v) sodium azide.

?

Use 1 ml of settled volume of resin coupled to E. coli antigen for each milligram of IgG protein to be

purified by affinity chromatography. Mix the IgG and the coupled resin and incubate for 12-18 hours

at room temperature on a rotating wheel device.

Load the slurry into a chromatography column. Recover the antibody by washing the column with TBS.

Collect fractions (0.2 column volume each) until the OD280 drops to zero. Pool the fractions containing

antibody, and store the pool at -20°

C until it is used for immunological screening.

REFERENCES

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1.

de

Wet,

J.R.,

Fukushima,

H.,

Dewji,

N.N.,

Wilcox,

E.,

O'Brien,

J.S.,

and

Helinski,

D.R.

1984.

Chromogenic immunodetection of human serum albumin and {alpha}-L-fucosidase clones in a human

hepatoma cDNA expression library. DNA 3: 437-447.[Medline]

Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory

makes no representations or warranties with respect to the material set forth in this protocol and has no

liability in connection with the use of these materials. Materials used in this protocol may be considered

hazardous and should be used with caution. For a full listing of cautions regarding these material, please

consult:

Molecular Cloning: A Laboratory Manual, Third Edition

, Joseph Sambrook and David W. Russell, ? 2001

by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p.14.28-14.30.

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GST

融合蛋白纯化方法

1

目的片 段接入

pGEX

载体；

2

涂板，挑单克隆，摇菌至

OD60 0≈1.0，加入

IPTG

（终浓度

1 mM

）诱导

6

－

8 h

；

3

收菌，每升菌液约以

50

mL PBS

重悬，加入

1%Triton

X-100

（

v/v

），

1

％

β

-

< br>巯基乙醇（

v/v

），

PMSF

（终浓度

1 mM

）；

以下步骤均在冰上操作：

4

超声破碎菌体，

15000 g

，

10min

离心取上清，在上清中加入适量

GST- beads

，轻轻晃动令其吸

附蛋白

1 h

；

5

2000 g

，

3min

离心弃上清；

6

加入至 少

10

倍体积

PBS

< br>，轻摇至

beads

悬浮于溶液中，

2000 g

，

3 min

离心弃上清；

7

重复步骤

6

两次；

8

加入

1 mL GST Elution Buffer

，轻摇

10 min

；

9

2000 g

，

3 min

离心，收集上清；

10

重复步骤

8-9

至少两次；