GST蛋白表达、纯化步骤

Protein Expression Protocol:

1.Transform control construct and tag-fusion-constructs into E.coli BL21(DE3) competent cell, grow

overnight;

2.Inoculate single colony to 2~3 ml LB medium, and grow at 37 degree for 7~10 hours or overnight;

3.1:100 inoculate to 3 ml LB, bacteria grow for 2-3 hours at 37 degree, then follow 1:2000 add 1 M

IPTG to induce protein expression overnight at 22 degree;

4.Spin down bacteria at 12000 rpm for 1 min, wash with 1 ml ddH2O, add 100 ul 1x PBS to

resuspend the deposition, then sonicate 3~5 min on ice;

5. Spin down at 12000 rpm for 10min, separate the supernatants and deposition;

6. Boiling with loading buffer at 100 degree for 5min, Spin down at 12000 rpm for 5min, 12%

SDS-PAGE;

7. After confirmed protein expressed in supernatants, increase the volume of bacteria medium. 1:100

inoculate to 100 ml LB, grow bacteria for 2-3 hours at 37 degree;then follow 1:2000 add 1 M IPTG to induce protein expression overnight at 22 degree;

8. Spin down bacteria at 8000 rpm 4 degree for 5 min, wash with 10 ml ddH2O, add 5 ml 1x PBS to

resuspend the deposition, then sonicate 60 min on ice;

9. Spin down at 12000 rpm 4 degree for 10min, separate the supernatants and deposition;

10.Prepare the supernatants for purification.

蛋白表达注意事项：

1. E.coli BL21比DH5α生长要快，固体培养基上10～12小时即可长斑，液体培养基中8小时后即可生长成

较大密度；切勿培养时间过长，防止菌体老化；

2.为了后续实验的便利，应尽量减少包涵体的形成。主要有以下方法可以减少包涵体的形成:

a.降低IPTG的浓度。IPTG终浓度0.2～0.8μM都可以诱导细菌表达蛋白；

b.加入IPTG后，把细菌生长温度降至16～30度。较低的生长温度降低了无活性聚集体形成的速率和疏水

相互作用，从而可减少包涵体的形成；

c.添加可促进重组蛋白质可溶性表达的添加剂，培养E.coli时添加高浓度的多醇类、蔗糖或非代谢糖可以阻

止分泌到周质的蛋白质聚集反应，在最适浓度范围内添加这些添加剂不会影响细胞的生长、蛋白质的合成或运输，其它添加剂还有乙醇（诱导热休克蛋白的表达）、低分子量的巯基或二硫化合物（影响细胞周质的还原态，从而影响二硫键的形成）和NaCl；

d.供给丰富的培养基，优化培养条件，如供氧、pH等。

3.超声后加入终浓度1% Triton x-100处理30～60min，有利于去除膜碎片和膜蛋白；

4.若需表达的蛋白含稀有密码子较多，尝试更换E.c oli宿主菌株，如Rosseta。

5.若表达毒性蛋白，造成细菌死亡或者生长缓慢，可以使用pLysS 菌株。

GST fusion protein purification （Glutathione Sepharose 4B ）

Buffer preparation ：

Water and chemicals used for buffer preparation should be of high purity. We recommend filtering the buffers by passing them through a 0.45 μm filter before use.

Binding buffer: PBS, pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH 2PO 4,

pH 7.3)

Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

GST-x Purification Protocol

1. Determine the volume of Glutathione Sepharose 4B required for your purification. As the bind capacity of Glutathione Sepharose 4B is > 5 mg glutathione-S-transferase/ml medium, generally, 150 ul ~ 200 ul slurry for 100 ml LB bacteria lysate is enough;

2. Gently shake the bottle of Glutathione Sepharose 4B to resuspend the slurry;

3. Sediment the medium by centrifugation at 12 000× g for 1 min. Carefully decant the supernatant;

4. Wash the Glutathione Sepharose 4B by adding 5 slurry volumes Binding buffer. Invert to mix;

5. Sediment the medium by centrifugation at 12 000 × g for 1 min. Carefully decant the supernatant;

6. Repeat steps 3 and 4 twice;

7. Trannsfer the medium to a 15 ml tube, add the cell lysate to the prepared Glutathione Sepharose 4B

and

incubate overnight at 4℃. Use gentle agitation such as end-over-end rotation;

8.Sediment the medium by centrifugation at 6000 × g for 5 min. Carefully decant the supernatant and save it

for measuring the binding efficiency to the medium i.e. by SDS-PAGE;

9.Wash the Glutathione Sepharose 4B by adding 5 slurry volumes Binding buffer, transfer the medium to a 1.5

ml tube. Invert to mix;

10.Sediment the medium by centrifugation at 12000 × g for 1 min. Carefully decant the supernatant (= wash)

and save it for SDS-PAGE analysis;

11.Repeat steps 9 and 10 twice for a total of three washes;

12.Elute the bound protein by adding 0.5 slurry volume Elution buffer. Incubate at room temperature for 1 h.

Using gentle agitation such as end-over-end rotation;

13.Sediment the medium by centrifugation at 12000 × g for 1 min. Carefully decant the supernatant (= eluted

protein);

14.Repeat steps 7 and 8 twice for a total of three elutions. Check the three eluates separately for purified protein

and pool according to the results;

15.Wash the Glutathione Sepharose 4B by adding 5 slurry volumes Binding Buffer. Invert to mix;

16.Sediment the medium by centrifugation at 12000 × g for 1 min. Carefully decant the supernatant;

17.repeat steps 14 and 15 twice;

18.Store Glutathone Sepharose 4B at 4°C in 20% ethanol.

GST融合蛋白纯化注意事项：

1. Binding Buffer和Elution Buffer中加入1–10 mM DTT，可以提高蛋白纯度，但是会导致其产率降低；

2. GST融合蛋白不与Glutathione Sepharose 4B(简称4B)结合：

a. 超声太剧烈或时间过长会引起蛋白变性，导致蛋白不能与4B结合。需注意设置好超声仪的功率和间隔时

间；

b. 在细胞裂解和加Binding Buffer和Elution Buffer之前，加入终浓度1–10 mM DTT可以显著提高GST融合

蛋白的结合效率；

c. 由于4B在pH值低于6.5或高于8.0结合效率会降低，因此使用前需用pH6.5～8.0的Buffer如PBS进行

平衡；

d. 如果4B已经使用过几次，有必要进行重生或者改用新鲜的4B；

3. GST融合蛋白不能有效洗脱：

a. 洗脱时间不足；

b. 增大Elution Buffer的洗脱体积；

c. 加大Elution Buffer中还原型glutathione的浓度。Glutathione的推荐浓度是10 mM，若此浓度洗脱效果不是

很理想可以尝试50 mM Tris- HCl, 20–40 mM reduced glutathione, pH 8.0 的Elution Buffer；

d. 增大Elution Buffer的pH值。Elution Buffer的pH值调至pH 8–9可以提高洗脱效率而不需要增加glutathione

的浓度；

e. 增加Elution Buffer的离子强度。加入0.1–0.2 M NaCl能提高洗脱效率；

f. 改用新鲜配置的Elution Buffer；

g. Elution Buffer中加入非离子型变性剂。非特异性的疏水作用可能会阻碍GST融合蛋白从4B上增溶和洗脱。

加入0.1% Triton X-100 or 2% 和N-octylglucoside可以显著增加洗脱效率。

4. Glutathione Sepharose 4B重生。如果Glutathione Sepharose 4B 上积累了沉淀物，变性的蛋白或者其他非特

异性蛋白，导致结合效率降低甚至丧失结合能力。可以用以下方法进行重生：

a. 去除沉淀物或者变性蛋白：用6 M盐酸胍洗2个柱体积后，紧接着用pH 7.3的PBS洗5个柱体积；

b. 去除疏水性的化合物：用70%乙醇洗3～4个柱体积或者用1% Triton? X-100洗2个柱体积后，紧接着用pH 7.3的PBS洗5个柱体积。

5. 最好用新鲜纯化的蛋白用于后续实验，根据实际情况选用蛋白定量试剂盒；

6. 上述注意事项未提到的问题请查阅说明书。