Protein Expression Protocol:
1.Transform control construct and tag-fusion-constructs li 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;
construct用法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 Na2HPO4, 1.8 mM KH2PO4, 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. Careful l y 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 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 Bi ndi ng B uffer. Invert to mi x;
16.Sediment the medium by centrifugation at 12000 ×g for 1 min. Careful l y decant the supernatant;
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, pH8.0 的El uti on B uffer；
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。

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