重组合无细胞蛋白合成系统PUREfrex

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

该反应系统由蛋白、核糖体、氨基酸和、NTPs组成[1,2]。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,且无需担心高背景影响到下游应用。

本试剂盒是各组分经过纯化再重新组合而成,而非直接从大肠杆菌中提取,RNaseβ-半乳糖苷酶以及脂多糖(LPS)污染已受到严格控制。PUREfrex® 试剂盒中的所有蛋白组分都不带标签,因此目的蛋白可融合任意标签进行纯化和检测。

http://www.boppard.cn/upfiles/images/201902/2615511502121122.png

◆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

◆功能及应用

以下为PUREfrex ® 已经过实验验证的应用功能,如需进一步数据,请联系在线客服索取技术资料。

验证起始密码子后的密码子对合成量的极大影响

已知若改变合成蛋白的起始密码子后的2-6号氨基酸密码子,合成蛋白的量会有很大的差异。这是以曲妥珠单抗(商品名赫赛汀)的Fab重链(VH+CH1)为模型,制备56种不同的密码子模板,比较其合成量。

膜蛋白的合成与纯化

在带His tag的Nanodisc上合成膜蛋白,进行亲和纯化的步骤时,即可从反应产物中纯化膜蛋白。

改善由于His tag 引起的N端合成量降低

α-Synuclein的N端带有His tag时合成蛋白,根据His tag序列的不同,有时合成量会降低。合成量的降低,只需更改His tag的基因序列就可以改善。

带His tag蛋白的纯化方法

用PUREfrex® 1.0 和PUREfrex® 2.0试剂盒中含有的DHFR制备带有His tag的模板DNA作为对照,并展示了用Ni亲和柱纯化后的结果。

混合了L链和H链模板的Fab抗体的合成实例

Fab是轻链(LC)和重链(HC)在分子内形成二硫键并缔合变活性型。向PUREfrex® 2.0 反应液中添加DS supplement合成Fab抗体后的结果显示,合成了可以和抗原结合的活性Fab抗体。另外,通过优化轻链(LC)和重链(HC)的模板DNA的添加比例,活性型Fab抗体的回收量也上涨了。

模板DNA序列和蛋白合成量

轻链(LC)和重链(HC)形成的Fab中,为了提高重链(HC)的合成量,通过向碱基序列中导入沉默突变,并改变5’端处的9个氨基酸的GC含量,调查其对合成量的结果显示,AT含量越高,合成量就有可能越高。海报中的案例更短,揭示了5’端处的6个氨基酸的GC含量变化的效果。

 

PUREfrex®2.0 的合成能力

PUREfrex® 1.0 和PUREfrex® 2.0分别合成相同的蛋白,比较其合成量以及活性。

 

合成各种各样的蛋白

通过向PUREfrex®(#PF001)的反应体系中添加DNA(或mRNA)并进行反应,不仅可以合成原核生物来源的蛋白,也可以合成真核生物来源的蛋白。例如,合成大肠杆菌的DHFR时,一个试剂盒(500 μL)可以合成50 μg以上的DHFR。下图的电泳是反应产物。

proteins-PUREfrex-300x298.jpg

图注:

● GFP : Green fluorescent protein *

● DHFR : Dihydrofolate reductase

● GST : Glutathione S-transferase

● β-Gal : β-Galactosidase

● bR : Bacteriorhodopsin

● Luc : Luciferase *

● CS : Citrate synthase *

● MDH : Malate dehydrogenase *

● Her2d1 : Domain1 of Her2 *

合成scFv

添加DnaK Mix,使用PUREfrex® 以及PUREfrex® SS 合成scFv(single-chain Fv),检测合成产物的活性。

合成微溶蛋白

使用添加了GroE Mix的PUREfrex® 1.0,合成4类大肠杆菌蛋白(FadA、HemB、PepQ、PyrC)*1,检测合成产物的可溶性。

*1)FadA, HemB, PepQ, PyrC是大肠杆菌内作为GroE的底物被报道的蛋白

*1)(Ref; Fujiwara et al. (2010) EMBO J, 29, 1552-1564)

合成荧光素酶

使用添加了DnaK Mix的PUREfrex® 合成荧光素酶,检测合成产物的活性。

合成有SS键的蛋白

在不同浓度的大肠杆菌DsbC存在下合成含有复数的二硫键蛋白,vtPA(truncated version of tissue plasminogen activator)和AppA(大肠杆菌酸性磷酸酶),并比较它们的活性。

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 μLⅹ5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 μLⅹ5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

备注:

PUREfrex® 已升级到2.0,比第一代产品表达量更高,污染物水平更低;

PUREfrex® 2.1比2.0更适合二硫键的形成。

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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