蛋白质组学

GST融合蛋白表达与纯化的实验步骤与注意事项

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蛋白表达技术全攻略

GST表达融合蛋白
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.

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/&mu;l(100&mu;M),工作浓度:10pmol/&mu;l(10&mu;M),-20°C保存
5、PCR扩增YFG
模板:质粒10ng/&mu;l 稀释少量 -20°C保存
引物:10pmol/&mu;l(10&mu;M) -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)

  25&mu;l反应体系 50&mu;l反应体系
模板 1&mu;l 2&mu;l
上游引物 1&mu;l 2&mu;l
下游引物 1&mu;l 2&mu;l
2×Master Mix 12.5&mu;l 25&mu;l
去离子H2 O 9.5&mu;l 19&mu;l

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5&mu;l 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25&mu;l BL21+ 3&mu;l质粒(300-500ng),混匀(质粒&le;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300&mu;l 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100&mu;l菌液加入5ml LB(Ampr)(50倍稀释),37°C,250rpm,过夜(16 h)
(8)取500&mu;l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100&mu;l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100&mu;l作为诱导后对照,连同诱导前菌液,12000rpm,离心2min,加入50&mu;l 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25&mu;l上清,加入25&mu;l 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25&mu;l,加入25&mu;l 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,30&mu;l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/&mu;l,10&mu;l)
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20&mu;l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100&mu;l 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50&mu;l,加入50&mu;l 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50&mu;l,加入50&mu;l 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,30&mu;l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200&mu;l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(3-5&mu;g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30&mu;l作为input对照(input与Pull-down蛋白量约为1/10)
(7)其余溶液,加入20&mu;l 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1-15&mu;g 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25&mu;l 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   10&mu;l 1M DTT
1mM PMSF   34&mu;l 0.3M PMSF
inhibitor cocktail   10&mu;l inhibitor cocktail

配方二

  500ml贮存液 10ml工作液
200mM NaCl   10ml贮存液
25mM HEPES pH7.5  
2mM DTT   20&mu;l 1M DTT
1mM PMSF   10&mu;l 1M PMSF
inhibitor cocktail   10&mu;l inhibitor cocktail

TBS(含5mM MgCl2、1mM DTT)

  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

常用IP细胞裂解液

  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   100&mu;l 100mM PMSF
1mM NaF 1ml 0.5M NaF 20&mu;l 0.5M NaF
complete protease inhibitors    

cleared lysates were incubated for 1.5 h with 3 &mu;g immobilized GST or GST-32

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