重组合无细胞蛋白合成系统

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。

 

>>>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。

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）。

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。

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。

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

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

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？ Ａ:　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？ Ａ:　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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