PUREfrex® 定制试剂盒

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  • 相关资料
  • Q&A
  • 参考文献

PUREfrex® 定制试剂盒PUREfrex® 定制试剂盒



◆产品简介


PUREfrex® 是基于PURE system技术基础上的无细胞蛋白合成系统,该系统由参与大肠杆菌蛋白合成的经单独纯化的因子重构所得。该试剂盒试剂组分可根据客户需求进行定制。

本定制试剂盒可按照订单要求,在PUREfrex® 1.0或2.0基础上进行定制。

PUREfrex® 定制试剂盒

PUREfrex® 1.0组成成分


溶液Ⅰ(缓冲液、盐、底物)

HEPES-KOH缓冲液pH 7.6

谷氨酸钾(Potassium glutamate)

乙酸镁(Magnesium acetate)

亚精胺(Spermidine)

二硫苏糖醇(Dithiothreitol)

10-甲酰四氢叶酸(10-Formyl-tetrahydrofolate)

4种NTPs

20种氨基酸

磷酸肌酸(Creatine phoshate)

tRNA混合物

 


溶液Ⅱ(缓冲液、盐、底物)

20种氨酰tRNA合成酶 (ARS)

甲硫氨酰tRNA转甲酰基酶(MTF)

3种起始因子(IF1、IF2、IF3)

3种延伸因子(EF-Tu、EF-Ts、EF-G)

3种释放因子(RF1、RF2、RF3)

核糖体循环因子(RRF)

T7 RNA聚合酶

核苷二磷酸激酶(NDK)

腺苷酸激酶(ADK、肌激酶)

肌酸激酶(CK)

焦磷酸酶(PPiase)

 


溶液Ⅲ

核糖体

 


◆应用案例

(-)RF1

 通过琥珀抑制,合成如下所示的包含非天然氨基酸的蛋白。

(-)T7 RNA聚合酶

 用于检测其他RNA聚合酶性能,如大肠杆菌RNA聚合酶。

(-)特异性氨基酸和(或)

  20种氨酰tRNA合成酶

 合成非天然氨基酸或少于20个氨基酸的蛋白。

PUREfrex® 定制试剂盒

◆定制流程


1. 请求定制协议并检查使用条款和条件;

2. 询问符合您定制的试剂盒被开发出的可能性;

3. 签署协议;

4. 接收为您定制试剂盒的产品编码;

5. 通过该产品编码进行订购。

 


◆协议


定制试剂盒在GeneFrontier公司协议下出售。使用这款产品的权利受限。

 


◆使用条件


● 本定制试剂盒仅供内部研究使用,非临床用途;

● 本定制试剂盒作非商业用途使用,且不允许分发给任何第三方;

● 试剂不得超出使用目的进行修改;

● 如果使用本试剂盒中的材料或材料的一部分取得新发明,客户需立即通知GFC公司;

● 所有与定制试剂盒材料本身有关的新发明以及其使用权均属于GFC公司;

● 所有通过使用定制试剂盒中材料合成的新产品以及其拥有权均属于客户。

● 工业客户需要签订额外的“许可协议”方可作体外展示用途

 


◆技术支持


欢迎与GeneFrontier的中国代理商联系,以了解以下内容:

● 了解关于签订协议的问题;

● 所需混合溶液是否能够制备;

● 询问适合自身实验的定制试剂盒的方案;

 


◆要求报价


价格按照每种反应体积而定。

可接受订购的最少反应体积为1 mL,以毫升为单位出售。这与以试剂盒为单位订购不同。详情请向我们咨询。

产品编号 产品名称 产品规格 产品等级

重组无细胞蛋白合成系统 PUREfrex® 2.0

  • 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。本系统的突出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,添加了DS supplement的结果显示,存在

3.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,为二硫键形成创造理想环境。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

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

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150 μg。

 

Q: 是否可以合成大于100 kDa的蛋白?

A: 我们用该试剂盒合成了116 kDa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0.5 mM。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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产品编号 产品名称 产品规格 产品等级
GFK-PF201-0.25-EX PUREfrex® 2.0 1 KIT
GFK-PF201-0.25-5-EX PUREfrex® 2.0 1 KIT
GFK-PF213-0.25-EX PUREfrex® 2.1 1 KIT
GFK-PF213-0.25-5-EX PUREfrex® 2.1 1 KIT
GFK-PF003-0.5-EX DnaK Mix 1 KIT
GFK-PF004-0.5-EX GroE Mix 1 KIT
GFK-PF005-0.5-EX DS supplement 1 KIT