ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR,ACS Chemical Biology

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ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2016-11-10 00:00:00 , DOI: 10.1021/acschembio.6b00838
Apostolos Alissandratos ₁ , Karine Caron ₁ , Thomas D. Loan ₁ , James E. Hennessy ₁ , Christopher J. Easton ₁

Affiliation

E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

中文翻译：

用细胞裂解液进行ATP循环以进行酶催化的化学合成，蛋白质表达和PCR

在几种重要的生物技术应用中，大肠杆菌裂解物可有效催化乙酰磷酸盐驱动的ATP再生。该ATP再循环策略在酶催化的化学合成中的用途通过重组尿苷激酶在大肠杆菌中的裂解物将尿苷转化为UMP来说明。通过裂解物中天然存在的激酶通过连续的磷酸化将UMP进一步转化为UTP，且产率很高。胞嘧啶核苷和5-氟尿苷也可通过该系统得到相应的NMP和NTP。与裂解物的提取物处理的无细胞蛋白质表达也进行容易时，代替加入所需的NTPs，所有四个产生原位使用乙酰磷酸并依靠内源性激酶活性从NMP中分离得到。同样，dNMP可用于产生PCR中DNA合成所需的dNTP。这些廉价的替代方案展示了利用容易获得的细胞裂解液进行乙酰磷酸和ATP回收的潜力。

更新日期：2016-11-10

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