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，RNase、β-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。
◆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
用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。
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？
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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