PUREfrex??試劑盒是在東京大學(xué)的Takuya Ueda教授所發(fā)明的PUREsystem技術(shù)基礎(chǔ)上，新開發(fā)的一款重組合無細(xì)胞蛋白合成試劑盒。
該反應(yīng)系統(tǒng)由蛋白、核糖體、氨基酸和、NTPs組成[1,2]。進行蛋白表達僅需將編碼目標(biāo)蛋白的模板DNA或mRNA加入到反應(yīng)體系中，然后孵育2-4小時即可完成反應(yīng)，且無需擔(dān)心高背景影響到下游應(yīng)用。
本試劑盒是各組分經(jīng)過純化再重新組合而成，而非直接從大腸桿菌中提取，RNase、β-半乳糖苷酶以及脂多糖（LPS）污染已受到嚴(yán)格控制。PUREfrex??試劑盒中的所有蛋白組分都不帶標(biāo)簽，因此目的蛋白可融合任意標(biāo)簽進行純化和檢測。
◆特點
● 可以同時加入多種模板進行反應(yīng)，以合成Fab（帶二硫鍵）及多聚體等帶二級結(jié)構(gòu)的多肽
● 可合成活細(xì)胞難以合成的強毒性蛋白
● 可直接使用PCR產(chǎn)物來作為模板DNA
● 單位體積內(nèi)合成的蛋白量幾乎恒定，不隨反應(yīng)體積變化而產(chǎn)生顯著差異
● 操作簡便，僅需在37℃孵育數(shù)小時
● 可以合成帶標(biāo)簽的蛋白用于下游純化和檢測
● 產(chǎn)品經(jīng)優(yōu)化升級，合成量大大提高
◆功能及應(yīng)用
以下為PUREfrex???已經(jīng)過實驗驗證的應(yīng)用功能，如需進一步數(shù)據(jù)，請聯(lián)系在線客服索取技術(shù)資料。
驗證起始密碼子后的密碼子對合成量的極大影響
已知若改變合成蛋白的起始密碼子后的2-6號氨基酸密碼子，合成蛋白的量會有很大的差異。這是以曲妥珠單抗（商品名赫賽?。┑腇ab重鏈（VH+CH1）為模型，制備56種不同的密碼子模板，比較其合成量。
膜蛋白的合成與純化
在帶His tag的Nanodisc上合成膜蛋白，進行親和純化的步驟時，即可從反應(yīng)產(chǎn)物中純化膜蛋白。
改善由于His tag 引起的N端合成量降低
α-Synuclein的N端帶有His tag時合成蛋白，根據(jù)His tag序列的不同，有時合成量會降低。合成量的降低，只需更改His tag的基因序列就可以改善。
帶His tag蛋白的純化方法
用PUREfrex??1.0?和PUREfrex??2.0試劑盒中含有的DHFR制備帶有His tag的模板DNA作為對照，并展示了用Ni親和柱純化后的結(jié)果。
混合了L鏈和H鏈模板的Fab抗體的合成實例
Fab是輕鏈（LC）和重鏈（HC）在分子內(nèi)形成二硫鍵并締合變活性型。向PUREfrex??2.0?反應(yīng)液中添加DS supplement合成Fab抗體后的結(jié)果顯示，合成了可以和抗原結(jié)合的活性Fab抗體。另外，通過優(yōu)化輕鏈（LC）和重鏈（HC）的模板DNA的添加比例，活性型Fab抗體的回收量也上漲了。
模板DNA序列和蛋白合成量
輕鏈（LC）和重鏈（HC）形成的Fab中，為了提高重鏈（HC）的合成量，通過向堿基序列中導(dǎo)入沉默突變，并改變5’端處的9個氨基酸的GC含量，調(diào)查其對合成量的結(jié)果顯示，AT含量越高，合成量就有可能越高。海報中的案例更短，揭示了5’端處的6個氨基酸的GC含量變化的效果。
PUREfrex?2.0?的合成能力
用PUREfrex??1.0?和PUREfrex??2.0分別合成相同的蛋白，比較其合成量以及活性。
合成各種各樣的蛋白
通過向PUREfrex?（#PF001）的反應(yīng)體系中添加DNA（或mRNA）并進行反應(yīng)，不僅可以合成原核生物來源的蛋白，也可以合成真核生物來源的蛋白。例如，合成大腸桿菌的DHFR時，一個試劑盒（500 μL）可以合成50 μg以上的DHFR。下圖的電泳是反應(yīng)產(chǎn)物。
圖注：
● 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），檢測合成產(chǎn)物的活性。
合成微溶蛋白
使用添加了GroE Mix的PUREfrex??1.0，合成4類大腸桿菌蛋白（FadA、HemB、PepQ、PyrC）*1，檢測合成產(chǎn)物的可溶性。
*1）FadA, HemB, PepQ, PyrC是大腸桿菌內(nèi)作為GroE的底物被報道的蛋白
*1）(Ref; Fujiwara et al. (2010) EMBO J, 29, 1552-1564)
合成熒光素酶
使用添加了DnaK Mix的PUREfrex??合成熒光素酶，檢測合成產(chǎn)物的活性。
合成有SS鍵的蛋白
在不同濃度的大腸桿菌DsbC存在下合成含有復(fù)數(shù)的二硫鍵蛋白，vtPA（truncated version of tissue plasminogen activator）和AppA（大腸桿菌酸性磷酸酶），并比較它們的活性。
◆產(chǎn)品列表
| 產(chǎn)品編號 | 產(chǎn)品名稱 | 規(guī)格 | 備注信息 |
| GFK-PF201-0.25-EX | PUREfrex??2.0 | 1 kit | 供250 μL反應(yīng)使用 |
| GFK-PF201-0.25-5-EX | 1 kit | 供250 μLⅹ5次反應(yīng)使用 | |
| GFK-PF213-0.25-EX | PUREfrex??2.1 | 1 kit | 供250 μL反應(yīng)使用 |
| GFK-PF213-0.25-5-EX | 1 kit | 供250 μLⅹ5次反應(yīng)使用 | |
| GFK-PF003-0.5-EX | DnaK Mix | 1 kit | 供500 μL反應(yīng)使用 |
| GFK-PF004-0.5-EX | GroE Mix | 1 kit | 供500 μL反應(yīng)使用 |
| GFK-PF005-0.5-EX | DS supplement | 1 kit | 供500 μL反應(yīng)使用 |
備注：
PUREfrex??已升級到2.0，比第一代產(chǎn)品表達量更高，污染物水平更低；
PUREfrex??2.1比2.0更適合二硫鍵的形成。
PUREfrex™?Q&A
| Ｑ: 使用PUREfrex™?試劑盒是否可用于真核蛋白的合成？
Ａ: PUREfrex™ 是由E.coli的核糖體和翻譯因子組成的體外重組蛋白合成試劑盒，但也可以合成哺乳動物和植物的蛋白。目標(biāo)蛋白的合成效率? ? ? ? ?取決于編碼蛋白的核苷酸序列，比如GC含量，稀有密碼子的含量。
Ｑ: 使用PUREfrex™ 試劑盒可以合成多少蛋白？ Ａ: 這個取決于目標(biāo)蛋白。來自E.coli的二氫葉酸還原酶每毫升反應(yīng)液可合成150 μg。
Ｑ: 是否可以合成大于100 kDa的蛋白？ Ａ: 我們用該試劑盒合成了116 kDa的蛋白。
Ｑ: 是否可以推薦PUREfrex™ 的反應(yīng)條件？ Ａ: 推薦用該試劑盒在37℃反應(yīng)2~4小時。
Ｑ: 是否可以合成和純化標(biāo)簽蛋白？ Ａ: 可以使用任何標(biāo)簽，PUREfrex™ 試劑盒的所有蛋白成分都沒有用于純化或者檢測的標(biāo)簽。比如，合成后可用金屬螯合的樹脂純化帶有His 標(biāo)簽的目標(biāo)蛋白。
Ｑ: 合成蛋白是否經(jīng)糖基化或者磷酸化修飾？ Ａ: 不。不會發(fā)生翻譯后修飾，PUREfrex™ 試劑盒只是由翻譯因子組成。
Ｑ: PUREfrex™ 試劑盒是否含有分子伴侶？ Ａ: 不。PUREfrex™ 試劑盒不含有任何分子伴侶，但你可以添加分子伴侶，比如Hsp70。你可以自己制備。
Ｑ: 用PUREfrex™ 試劑盒是否可合成含有二硫鍵的蛋白？ Ａ: 不行。目標(biāo)蛋白合成不帶有二硫鍵，因為翻譯反應(yīng)時有還原劑DTT。大多數(shù)需要二硫鍵才有活性的蛋白，會沒有活性。
Ｑ: PUREfrex™ 是否可合成膜蛋白？ Ａ: 一般情況，合成膜蛋白會形成聚集。為了獲得能夠插入到脂雙層的膜蛋白，需要在合成膜蛋白時添加脂質(zhì)體到PUREfrex™。
Ｑ: 是否可合成帶有[35S] 甲硫氨酸或者 [3H] 亮氨酸的蛋白？ Ａ: 添加放射性元素標(biāo)記的氨基酸可以合成放射性元素標(biāo)記的蛋白，比如[35S] 甲硫氨酸或者 [3H] 亮氨酸。PUREfrex™ 含有20種天然的氨基 酸，濃度都在0.5 mM。請優(yōu)化條件。
Ｑ: 除了T7啟動子外，是否可用其他啟動子？ Ａ: 我們推薦使用T7啟動子的模板DNA，因為PUREfrex™ 含有轉(zhuǎn)錄的RNA聚合酶。當(dāng)你使用其他聚合酶，制備的模板DNA要有相應(yīng)聚合酶的 合適啟動子。
Ｑ: 使用DHFR DNA(陽性對照)無法獲得DHFR。 Ａ: 該試劑盒由于某些原因失活。為了避免失活，請將該試劑盒存放在適當(dāng)穩(wěn)定。可進行分裝，避免反復(fù)凍融影響試劑盒的使用效果?；蛘吒?/p> 試劑盒被核酸酶污染了。請使用不含核酸酶的水，試劑和材料。 Ｑ: 使用試劑盒的DHFR可以得到DHFR。但是不能得到目標(biāo)蛋白，或者目標(biāo)蛋白量很低。 Ａ: 1）改試劑盒由于某些原因失活了。為了避免失活，請將該試劑盒存放在適當(dāng)?shù)臏囟炔⑶疫M行分裝（避免反復(fù)凍融） Ａ: 2）可以受核酸酶污染。為了避免核酸酶污染，請使用不含核酸酶的水，試劑和材料。 Ａ: 3）制備的DNA模板不準(zhǔn)確。需要制備含有T7啟動子，核糖體結(jié)合位點，起始密碼子，終止密碼子的DNA模板。 Ａ: 4）轉(zhuǎn)錄的二級結(jié)構(gòu)會阻止翻譯反應(yīng)。這種情況，請優(yōu)化模板的順序，解決二級結(jié)構(gòu)的問題。 |
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