重组无细胞蛋白合成系统 PUREfrex® 2.0


产品编号 产品名称 产品规格 产品等级 产品价格
GFK-PF201-0.25-EX PUREfrex® 2.0 1KIT
GFK-PF201-0.25-5-EX PUREfrex® 2.0 1KIT
GFK-PF213-0.25-EX PUREfrex® 2.1 1KIT
GFK-PF213-0.25-5-EX PUREfrex® 2.1 1KIT
GFK-PF003-0.5-EX DnaK Mix 1KIT
GFK-PF004-0.5-EX GroE Mix 1KIT
GFK-PF005-0.5-EX DS supplement 1KIT
  • 产品特性
  • 相关资料
  • Q&A
  • 参考文献

重组合无细胞蛋白合成系统重组无细胞蛋白合成系统                  PUREfrex® 2.0

PUREfrex® 2.0

 



◆简介


  PUREfrex® 试剂盒是在东京大学的Takuya Ueda教授所发明的PUREsystem技术基础上,新开发的一款重组合无细胞蛋白合成试剂盒。

  反应系统由蛋白质、核糖体、氨基酸和NTPs组成[1,2],其中蛋白行使转录、翻译和能量供应的功能。蛋白与核糖体为分别单独高度纯化后,再重新组合成蛋白合成系统,而非直接从大肠杆菌S30中提取。当合成蛋白时,仅需将编码目的蛋白的模板DNA或mRNA添加到反应混合液中并孵育数小时,即可完成反应。本系统最大的特色,是在体外以转录相关因子重新组合一套表达系统,并可根据需要来调整反应混合物的成分,而不必担心高背景会影响下游的应用。进行蛋白表达仅需将编码目标蛋白的模板DNA或mRNA加入到反应体系中,然后孵育2-4小时即可完成反应。PUREfrex® 试剂盒的所有蛋白组分均不带标签,因此目的蛋白可融合任意标签进行纯化和检测。

重组无细胞蛋白合成系统                  PUREfrex® 2.0

 


>>>无细胞表达的优势<<<


● 无需制备克隆

● 无需考虑培养条件

● 无需考虑表达所需的诱导条件

● 来源于宿主的污染少

 



◆PUREfrex® 系列


● PUREfrex® 1.0 第一代产品    

● PUREfrex® 2.0 第二代产品,表达量更高,污染水平更低;

● RNA酶与β-半乳糖苷酶污染大大降低;

● 每1 µL反应混合物中的脂多糖(LPS)低于0.1 EU。

● PUREfrex® 2.1 更适合二硫键的形成



☆升级至PUREfrex® 2.0


1.合成原核和真核蛋白的结果显示,用PUREfrex® 2.0合成时,各种蛋白的合成量增加。

重组无细胞蛋白合成系统                  PUREfrex® 2.0

2.GFP蛋白合成的结果显示,用PUREfrex® 2.0合成时,可以观察到荧光强度增强了

2.10倍以上(每单位反应产物)。

重组无细胞蛋白合成系统                  PUREfrex® 2.0

3.合成需要形成二硫键(SS键)的大肠杆菌酸性磷酸酶(AppA1)时,在PUREfrex® 2.0基础上,

3.添加了DS supplement的结果显示,存在氧化剂和二硫键异构酶时,活性蛋白合成量增加。

重组无细胞蛋白合成系统                  PUREfrex® 2.0

1:AppA有5个二硫键,是其中一个位点在不连续的半胱氨酸之间存在的二硫键。



4.正确高级结构蛋白的合成结果显示,存在分子伴侣2的情况下用PUREfrex® 2.0,蛋白合成量增加。

重组无细胞蛋白合成系统                  PUREfrex® 2.0

2 DnaK Mix:DnaK / DnaJ / GrpE mixture为配套分子伴侣

 



◆特点


● 可以同时加入多种模板进行反应,以合成Fab(带二硫键)及多聚体等带二级结构的多肽

● 可合成活细胞难以合成的强毒性蛋白

● 可直接使用PCR产物来作为模板DNA

● 单位体积内合成的蛋白量几乎恒定,不随反应体积变化而产生显著差异

● 操作简便,仅需在37℃孵育数小时

● 可以合成带标签的蛋白用于下游纯化和检测

● 产品经优化升级,合成量大大提高

 



◆应用


制备目的蛋白


● 原核蛋白

● 真核蛋白

● 膜蛋白

● 二硫键蛋白

● 含有非天然氨基酸的蛋白质等

 


蛋白基础研究


● 翻译

● 蛋白合成后折叠

 


体外展示技术


● 核糖体展示技术

● mRNA展示技术

 



◆应用实例


利用PUREfrex® 系统合成并一步纯化DHFR-His

重组无细胞蛋白合成系统                  PUREfrex® 2.0

3:模板DNA的构建方法请见"相关资料"栏或点击这里




◆添加剂(用于需要形成二硫键和分子伴侣的蛋白质)


● DS supplement

● 通过添加DS supplement至PUREfrex® 反应液中,为二硫键形成创造最佳的环境。DS supplement作为创造氧化环境的氧化剂,含有氧化型谷胱甘肽(GSSG)和作为二硫键异构酶的大肠杆菌DsbC。当蛋白需要二硫键才能产生活性形式时,请使用本添加剂。

 

● DnaK Mix

● DnaK Mix是高度纯化后的大肠杆菌来源的DnaK、DnaJ、GrpE蛋白以适当的浓度比例预混后的溶液。在PUREfrex® 反应体系中单独或添加DS supplement合成蛋白时同时添加DnaK Mix,可以更易获得难以独自形成高级结构的活性蛋白。

 

● GroE Mix

● GroE Mix是高度纯化后的大肠杆菌来源的GroEL、GroES蛋白以适当的浓度比例预混后的溶液。以PUREfrex® 反应体系合成蛋白时添加GroE Mix,可以更易获得难以独自形成高级结构的活性蛋白。

 



◆试剂盒组成


用于250 μL反应

使用前请将试剂盒置于-80°C保存

试剂 体积 成分说明 保存温度
溶液 I (白盖 125 μL 氨基酸,核苷酸,tRNA和酶的底物等 -20°C
溶液 II (黑盖 12.5 μL 蛋白,保存于含30%甘油的缓冲液 -20°C or -80°C(1)
溶液 III (红盖 12.5 μL ×2 核糖体(20 μM) -80°C(1)

DHFR   DNA (透明盖)(2)

10 μL 对照DNA,含有编码大肠杆菌DHFR基因的PCR产物(20 ng/μL) -20°C

(1)剩余的溶液应快速在液氮、干冰或乙醇中冻结,并储存于-80℃。如有必要,分装剩余溶液,并尽可能避免反复冻融。

(2)每50 μL反应中加入2.5 μL DHFR DNA。

 

◆产品列表


产品编号

产品名称

规格

备注信息

GFK-PF201-0.25-EX

PUREfrex® 2.0

1 kit

供250 μL反应使用

GFK-PF201-0.25-5-EX

PUREfrex® 2.0

1 kit

供250 μL×5次反应使用

GFK-PF213-0.25-EX

PUREfrex® 2.1

1 kit

供250 μL反应使用

GFK-PF213-0.25-5-EX

PUREfrex® 2.1

1 kit

供250 μL×5次反应使用

GFK-PF003-0.5-EX

DnaK Mix

1 kit

供500 μL反应使用

GFK-PF004-0.5-EX

GroE Mix

1 kit

供500 μL反应使用

GFK-PF005-0.5-EX

DS supplement

1 kit

供500 μL反应使用

 

相关产品的详细信息请点击查看:重组无细胞蛋白合成系统 PUREfrex® 2.0

相关资料

重组无细胞蛋白合成系统                  PUREfrex® 2.0 重组无细胞蛋白合成系统                  PUREfrex® 2.0

PUREfrex™ Technical information

PUREfrex™ Protocol

蛋白质工程相关产品

PUREfrex:重组无细胞蛋白合成试剂盒

RYTS试剂盒:大肠杆菌无细胞蛋白质合成系统

CloverDirect:定点蛋白质功能化tRNA试剂

纯化系统:一步高纯度标记纯化系统

STELLA +“赖氨酸标记试剂盒”

  • PUREfrex : Reconstituted Cell-free Protein Synthesis Kit

  • RYTS Kit : E. coli Cell-free Protein Synthesis System

  • CloverDirect : tRNA Reagents for Site-Directed Protein Functionalization

  • Dock Purification System : One step high purity purification tag purification system

  • STELLA+ " Lysine Labeling Kit "

PUREfrex Q&A

Q: 使用PUREfrex™ 试剂盒是否可用于真核蛋白的合成?

: PUREfrex™ 是由E.coli的核糖体和翻译因子组成的体外重组蛋白合成试剂盒,但也可以合成哺乳动物和植物的蛋白。目标蛋白的合成效率取决于编码蛋白的核苷酸序列,比如GC含量,稀有密码子的含量。

 

Q: 使用PUREfrex™ 试剂盒可以合成多少蛋白?

: 这个取决于目标蛋白。来自E.coli的二氢叶酸还原酶每毫升反应液可合成150ug。

 

Q: 是否可以合成大于100kDa的蛋白?

A: 我们用该试剂盒合成了116kDa的蛋白。

 

Q: 是否可以推荐PUREfrex™ 的反应条件?

A: 推荐用该试剂盒在37℃反应2~4小时。

 

Q: 是否可以合成和纯化标签蛋白?

A: 可以使用任何标签,PUREfrex™ 试剂盒的所有蛋白成分都没有用于纯化或者检测的标签。比如,合成后可用金属螯合的树脂纯化带有His标签的目标蛋白。

 

Q: 合成蛋白是否经糖基化或者磷酸化修饰?

A: 不。不会发生翻译后修饰,PUREfrex™ 试剂盒只是由翻译因子组成。

 

Q: PUREfrex™ 试剂盒是否含有分子伴侣?

A: 不。PUREfrex™ 试剂盒不含有任何分子伴侣,但你可以添加分子伴侣,比如Hsp70。你可以自己制备。

 

Q: 用PUREfrex™ 试剂盒是否可合成含有二硫键的蛋白?

A: 不行。目标蛋白合成不带有二硫键,因为翻译反应时有还原剂DTT。大多数需要二硫键才有活性的蛋白,会没有活性。

 

Q: PUREfrex™ 是否可合成膜蛋白?

A: 一般情况,合成膜蛋白会形成聚集。为了获得能够插入到脂双层的膜蛋白,需要在合成膜蛋白时添加脂质体到PUREfrex™。

 

Q: 是否可合成带有[35S] 甲硫氨酸或者 [3H] 亮氨酸的蛋白?

A: 添加放射性元素标记的氨基酸可以合成放射性元素标记的蛋白,比如[35S] 甲硫氨酸或者 [3H] 亮氨酸。PUREfrex™ 含有20种天然的氨基酸,浓度都在0.5mM。请优化条件。

 

Q: 除了T7启动子外,是否可用其他启动子?

A: 我们推荐使用T7启动子的模板DNA,因为PUREfrex™ 含有转录的RNA聚合酶。当你使用其他聚合酶,制备的模板DNA要有相应聚合酶的合适启动子。

 

Q: 使用DHFR DNA(阳性对照)无法获得DHFR。

A: 该试剂盒由于某些原因失活。为了避免失活,请将该试剂盒存放在适当稳定。可进行分装,避免反复冻融影响试剂盒的使用效果。或者改试剂盒被核酸酶污染了。请使用不含核酸酶的水,试剂和材料。

 

Q: 使用试剂盒的DHFR可以得到DHFR。但是不能得到目标蛋白,或者目标蛋白量很低。

A: 1)改试剂盒由于某些原因失活了。为了避免失活,请将该试剂盒存放在适当的温度并且进行分装(避免反复冻融)

A: 2)可以受核酸酶污染。为了避免核酸酶污染,请使用不含核酸酶的水,试剂和材料。

A: 3)制备的DNA模板不准确。需要制备含有T7启动子,核糖体结合位点,起始密码子,终止密码子的DNA模板。

A: 4)转录的二级结构会阻止翻译反应。这种情况,请优化模板的顺序,解决二级结构的问题。

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