GST融合蛋白纯化方法

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OD600≈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-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，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；

92000 g，3 min离心，收集上清；

10重复步骤8-9至少两次；

11 SDS-PAGE电泳检测蛋白纯度，Bradford法检测蛋白浓度；

12将蛋白置于-20℃保存。

P.S. 大量提取前应取少量菌液，改变IPTG浓度，诱导温度，诱导时间等，以确定蛋白表达的最适条

Thrombin cleavage

(using thrombin produced by Amersham)

1. Thrombin cleavage of eluted fusion protein bound to Sepharose

* Mix 50 μl of thrombin (< 10 cleavage U/ml) solution and 950 μl of 1 x PBS for each ml of Glutathione Sepharose bed volume

Add thrombin protease mixture to Glutathione Sepharose pellet

Gently shake or rotate the suspension at r.t. for 2-16 h

Centrifuge the suspension at 500 x g for 5’ to pellet the beads and carefully transfer the eluted fraction to a clean tube.

2. Thrombin cleavage of eluted fusion protein.

Add 10 μl of thrombin solution (10 cleavage units) per mg fusion protein. If the amount of fusion protein in the eluate has not been determined, add 80 μl (80 U) of thrombin for each ml of Glutathione Sepharose bed volume from with the fusion protein was eluted.

Mix gently and incubate at r.t. (22-25C) for 2-16 h.

Once digestion is complete, GST can be removed by first removing glutathione by extensive dialysis (2,000 vol/ml) against 1 x PBS followed by batch purification.