GST融合蛋白表达与纯化的实验步骤与注意事项
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载体
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. |
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 |
(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
7、转化BL21(DE3)pLysS菌株检测GST融合蛋白的表达
(1)冰上融化BL21(DE3)pLysS感受态细胞(天根)
(2)2 ml离心管中,加入25μl BL21+ 3μl质粒(300-500ng),混匀(质粒≤感受态1/10)
(3)冰上放置30min
(4)42°C,90s
(5)冰上放置2-3min
(6)加入300μl LB(无抗生素)(相当于菌体10倍体积的LB),37°C,250rpm,1 h
(7)4000rpm,2min,弃上清,其余混匀以后涂平板(Ampr),37°C,过夜(16 h)
(8)挑单克隆菌落,5ml LB(Ampr),37°C,250rpm,过夜(16 h)
()稀释10倍、20倍、50倍测定OD600
()选择合适的稀释倍数,5ml菌液,37°C,250 rpm,1 h,测定OD600
(7)取100μl菌液加入5ml LB(Ampr)(50倍稀释),37°C,250rpm,过夜(16 h)
(8)取500μl菌液加入5ml LB(Ampr)(10倍稀释)(其余菌液4°C保存),测OD600
(9)37°C,250 rpm,2 h(OD600:0.6-0.8),测OD600
(10)室温放置20 min(摇床降温,30°C)
(11)取100μl作为诱导前对照,冰上放置
(12)菌液中加入IPTG,终浓度0.5-1 mM
(13)30°C,250 rpm,2 -4h(可做时间梯度),测OD600
(14)取100μl作为诱导后对照,连同诱导前菌液,12000rpm,离心2min,加入50μl 1×SDS上样缓冲液
(15)取4ml菌液,12000rpm,离心2min,收集到2ml离心管中
(16)加入1ml GST裂解缓冲液重悬菌体
(17)超声破碎细胞:超声20s,间隔10s,共3-5次,超声强度200-300W(冰浴)
(18)超声后菌液换入一个新1.5ml离心管,4°C,12000rpm,离心5min
(19)超声后上清:取25μl上清,加入25μl 2×SDS上样缓冲液(其余上清-80°C保存)
(20)超声后沉淀:沉淀加入1ml GST裂解缓冲液重悬,取25μl,加入25μl 2×SDS上样缓冲液(其余沉淀-80°C保存)
(21)将四个样品煮沸3min,12000rpm,离心5min
(22)8-10%SDS-PAGE,30μl上样,顺序:诱导前菌体、诱导后菌体、超声后上清、超声后沉淀
(23)考马斯亮蓝染色
[注意]
(1)菌液OD值小于1即可
(2)诱导时间最好做一个梯度,2-6h
(3)诱导温度适当摸索,24°C、30°C
(4)IPTG浓度可做梯度,1mM
(5)超声条件可视实际情况改变,只要使细菌裂解充分即可,即菌液清亮不粘稠
8、测序确认
上海生工生物工程技术服务有限公司(Tel:81767529)
测序引物:pGEX-3通用引物
测序样品:过夜菌1ml;质粒(浓度大于50ng/μl,10μl)
9、中提质粒(Promega)
10、表达纯化GST融合蛋白
(1)已经转化的菌液,或者重新转化BL21(DE3)pLysS
(2)活化:取20μl菌液加入11ml LB(Ampr)(500倍稀释),37°C,250rpm,过夜(16 h)
(3)取10ml菌液加入100ml LB(Ampr)(10倍稀释)(剩余菌液4°C保存)
(4)37°C,250 rpm,1 h 10 min-1 h 20 min,OD600 0.6-0.8(OD600小于1.0即可)
(5)取1ml菌液作为诱导前对照,冰上放置
(6)加入IPTG,终浓度1 mM
(7)30°C,250rpm,诱导适当时间(预实验确定)
(8)取1ml菌液作为诱导后对照,连同诱导前菌液,12000rpm,离心2min,收集菌体,加入100μl 1×SDS上样缓冲液
(9)其余菌液,分为两份,倒入50ml离心管,5000rpm,离心20 min,收集细胞
(10)每管加入8-10ml GST裂解缓冲液重悬菌体,转移小烧杯中(无气泡)
(11)超声破碎细胞:超声3s,间隔10s,40次左右,超声强度200-300W(冰浴)
(12)将超声后菌液转移到50ml离心管中,4°C,13000rpm,离心20-30min
(13)将上清转移到1个50ml离心管中,取出50μl,加入50μl 2×SDS上样缓冲液(其余上清-80°C保存)
(14)将沉淀加入16-20ml GST裂解缓冲液(或PBS)重悬,取出50μl,加入50μl 2×SDS上样缓冲液(其余沉淀-80°C保存)
(15)将四个样品煮沸3min,12000rpm,离心5min
(16)8-10%SDS-PAGE检测诱导、超声是否合适,30μl上样,顺序:诱导前菌体、诱导后菌体、超声后上清、超声后沉淀
(17)考马斯亮蓝染色
(18)混匀glutathione sepharose beads(4B)(mixed slurry),取200μl加入15ml离心管中(2份)
(19)加入10ml PBS,混匀,3000rpm,离心3min,弃上清
(20)加入超声后上清液,4°C轻柔摇荡60分钟(或过夜)(横放)
(21)4°C,3000rpm,离心3min
(22)10ml GST裂解缓冲液洗涤2次(冰上)
(23)10ml TBS(含5mM MgCl2、1mM DTT)洗涤2次(冰上)
(24)弃上清,加入1倍柱床体积的TBS(含5mM MgCl2、1mM DTT、50%甘油),混匀
(25)取悬液测定蛋白浓度或SDS-PAGE
(26)-20°C保存beads
11、GST-Pull-down
(1)10cm培养皿中,未处理的细胞或者转染的细胞(3-5μg质粒转染10cm培养皿,Cos-7、293T或者其他细胞,转染24h)
(2)加入1ml 细胞裂解缓冲液,细胞铲刮下细胞(冰上)
(3)将裂解液收集到1.5ml离心管中,振荡30s,冰上放置5min,重复2-3次,充分裂解细胞
(4)4°C,12000rpm,离心15min,收集上清
(5)测定蛋白浓度,取1mg总蛋白做GST-Pull-down?
(6)留30μl作为input对照(input与Pull-down蛋白量约为1/10)
(7)其余溶液,加入20μl glutathione sepharose beads(PBS洗涤过),4°C,轻柔振荡20min
(8)12000rpm,离心3min
(9)收集上清,分为两份,一份加入1-15μg GST结合的beads,另一份加入等量 GST-融合蛋白结合的beads,4°C,轻柔振荡60min
(10)3000rpm,离心3min,回收上清
(11)1ml 细胞裂解缓冲液洗涤3次
(12)弃尽上清,加入25μl 2×SDS上样缓冲液
(13)煮沸3min,12000rpm,离心3min
(14)SDS-PAGE,上样顺序:细胞裂解液input、x、GST、x、GST-融合蛋白(x:LSB)
(15)考马斯亮蓝染色或免疫印迹
[附录]
GST裂解缓冲液(前四个组分配好之后4°C保存,DTT和蛋白酶抑制剂现用现加)
配方一
500ml贮存液 | 10ml工作液 | |
150mM NaCl | 15 ml 5M NaCl | 10ml贮存液 |
20mM HEPES pH7.5 | 20ml 0.5M Hepes pH7.5 | |
5mM MgCl2 | 10ml 1M MgCl2 | |
1% TritonX-100 | 5ml 100% Triton-X 100 | |
1mM DTT | 10μl 1M DTT | |
1mM PMSF | 34μl 0.3M PMSF | |
inhibitor cocktail | 10μl inhibitor cocktail |
500ml贮存液 | 10ml工作液 | |
200mM NaCl | 10ml贮存液 | |
25mM HEPES pH7.5 | ||
2mM DTT | 20μl 1M DTT | |
1mM PMSF | 10μl 1M PMSF | |
inhibitor cocktail | 10μl inhibitor cocktail |
500ml 贮存液 | ||
150mM NaCl | 15ml 5M NaCl | |
20mM Tris pH7.6 | 10ml 1M Tris pH7.6 | |
5mM MgCl2 | 0.5M MgCl2 | |
1mM DTT | 1M DTT |
500ml贮存液 | 10ml 工作液 | |
50mM Tris-HCl, pH7.5 | 25ml 1M Tris-HCl, pH7.5 | |
150mM NaCl | 15ml 5M NaCl | |
2mM EDTA | 2ml 0.5M EDTA | |
0.5% NP40 | 25ml 10% NP40 | |
0.5% Triton X-100 | 25ml 10% Triton X-100 | |
0.5mM DTT | 2.5ml 1M DTT | |
1mM PMSF | 100μl 100mM PMSF | |
1mM NaF | 1ml 0.5M NaF | 20μl 0.5M NaF |
complete protease inhibitors |