GST融合蛋白表达与纯化的实验步骤与注意事项（一）
GST表达融合蛋白
pGEX-KG
大小：5006bp，氨苄青霉素抗性(Ampr)，IPTG诱导表达
酶切位点：BamHI 930、SmaI 937、EcoRI 962、XbaI 966、NcoI 974、SalI 980、XhoI 985、SacI 992、HindIII 994
GST分子量：
构建pGEX-KG-YFG重组 质粒
1、分析所感兴趣的基因(your favorite gene, YFG)
Primer Premier 5.0软件，分析YFG含有哪些酶切位点，注意是否与pGEX-KG载体 的多克隆位点有重合
2、确定合适的双酶切位点
NEB网站(www.neb.com) Double Digest Finder软件 ，查找最佳双酶切组合(下表)
| NEB 双酶切图谱 | |||
| BamHI | EcoRI | NEBuffer EcoRI + BSA at 37°C. | BamHI may exhibit star activity in this buffer. |
| XbaI | NEBuffer 3 + BSA at 37°C. | At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. | |
| NcoI | NEBuffer 3 + BSA at 37°C. | ||
| SalI | NEBuffer 3 + BSA at 37°C | ||
| XhoI | NEBuffer 3 + BSA at 37°C. | ||
| SmaI | XbaI | NEBuffer 4 + BSA at 25°C with SmaI, then add XbaI and raise temperature to 37°C. | At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. |
| NcoI | NEBuffer 4 at 25°C with SmaI, then add NcoI and raise temperature to 37°C. | ||
| XhoI | NEBuffer 4 + BSA at 25°C with SmaI, then add XhoI and raise temperature to 37°C. | ||
| SacI | NEBuffer 4 + BSA at 25°C with SmaI, then add SacI and raise temperature to 37°C. | ||
| HindIII | NEBuffer 4 at 25°C with SmaI, then add HindIII and raise temperature to 37°C. | At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. | |
| EcoRI | NcoI | NEBuffer EcoRI at 37°C. | |
| SalI | NEBuffer EcoRI + BSA at 37°C. | ||
| XhoI | NEBuffer EcoRI + BSA at 37°C. | ||
| SacI | NEBuffer 1 + BSA at 37°C. | EcoRI may exhibit star activity in this buffer. | |
| HindIII | NEBuffer EcoRI at 37°C. | ||
| XbaI | NcoI | NEBuffer 2 + BSA at 37°C. | |
| SalI | NEBuffer 3 + BSA at 37°C. | At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. | |
| XhoI | NEBuffer 2 + BSA at 37°C. | ||
| SacI | NEBuffer 4 + BSA at 37°C. | This buffer is not supplied with either enzyme. At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. |
|
| HindIII | NEBuffer 2 + BSA at 37°C. | ||
| NcoI | SalI | NEBuffer 3 + BSA at 37°C. | |
| XhoI | NEBuffer 2 + BSA at 37°C. | ||
| SacI | NEBuffer 1 + BSA at 37°C. | ||
| HindIII | NEBuffer 2 at 37°C. | ||
| SalI | XhoI | NEBuffer 3 + BSA at 37°C. | |
| XhoI | SacI | NEBuffer 1 + BSA at 37°C. | At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. |
| HindIII | NEBuffer 2 + BSA at 37°C. | ||
| SacI | HindIII | NEBuffer 2 + BSA at 37°C. | At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary. |
对照YFG、载体多克隆位点，确定上、下游酶切位点
3、设计PCR上、下游引物
Primer Premier 5.0软件，设计PCR上、下游引物
酶切位点外最多含6个碱基
3’端不是A，最好是G或C，但是不推荐使用GC或CG结尾
3’端至少保证有10个碱基完全配对
得分(Rating)大于70
[注意]
上游引物：是否添加适当碱基，确保不打乱开放阅读框
下游引物：添加终止密码子(UAA、UAG、UGA)
4、引物合成及保存
合成：上海生工生物工程技术服务有限公司(Email：beijing@sangon.com，Tel：81767586);纯化方法：柱层析or聚丙烯酰胺凝胶电泳?;价格1.30/碱基
保存：贮存浓度：100pmol/μl(100μM)，工作浓度：10pmol/μl(10μM)，-20°C保存
5、PCR扩增YFG
模板：质粒10ng/μl 稀释少量 -20°C保存
引物：10pmol/μl(10μM) -20°C保存
Taq酶：NEB Quick-Load Taq 2×Master Mix 扩增片段小于2.0kb
反应体系(配制时置于冰上)
| 25μl反应体系 | 50μl反应体系 | |
| 模板 | 1μl | 2μl |
| 上游引物 | 1μl | 2μl |
| 下游引物 | 1μl | 2μl |
| 2×Master Mix | 12.5μl | 25μl |
| 去离子H2 O | 9.5μl | 19μl |
反应条件
(1) 预变性 94°C 5 min
(2) 变性 94°C 30 s
(3) 退火 待定 30 s
(4) 延伸 72°C 待定
(5) 重复2-5 25-30个循环
(6) 补平缺口 72°C 10 min
(7) 暂存 10°C
[注意]
退火温度：参考4(G+C)+2(A+T)-4(互补碱基)，参考Ta Opt(Primer Premier 5.0)
延伸时间：Taq酶：1kb/min
循环数小于30，减少错配
琼脂糖电泳检测PCR产物
0.8%有效分离范围：10~0.8kb;1.0%有效分离浓度7~0.5kb
50ml TAE加入5μl EB母液(5mg/ml)
100V，30-45min
拍照或者紫外灯下切胶回收
6、构建pGEX-KG-YFG
酶切：双酶切PCR产物、pGEX-KG
回收：PCR产物直接回收、pGEX-KG电泳之后切胶回收
连接：pGEX-KG 50ng、插入片段150ng
转化铺平板：Ampr
挑单克隆：Ampr(四个菌落足够了)
鉴定：小提质粒酶切 or菌体 PCR
