Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coli.
Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coli.
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DOI：
10.1111/j.1751-7915.2008.00041.x
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发表时间：
2008-09
影响因子：
5.7
通讯作者：
DeLisa MP
中科院分区：
文献类型：
--
作者：
Fisher AC;Kim JY;Perez-Rodriguez R;Tullman-Ercek D;Fish WR;Henderson LA;DeLisa MP
历史上，革兰氏阴性菌的一般分泌（Sec）途径在许多表达和工程应用中充当了将异源蛋白递送至周质的主要途径。在这里，我们系统地研究了双精氨酸易位（达特）途径作为一种替代，可能是有利的，异源蛋白质的分泌途径。总体而言，我们发现：（i）模型底物的输出效率和周质产量受达特信号肽的组成影响，（ii）达特底物在到达周质时在其N末端被正确加工，和（iii）与麦芽糖结合蛋白（MBP）融合的蛋白质通过达特系统可靠地输出，但仅当正确折叠时;通过达特途径的折叠质量控制特征排除异常折叠的MBP融合体。我们还观察到，对于相对小的折叠良好的蛋白质，达特输出产率与Sec相当，对于需要细胞质折叠的蛋白质，输出产率相对于Sec较高，对于较大的可溶性融合蛋白质，输出产率相对于Sec较低。有趣的是，从周质纯化的材料的比活性对于某些达特底物相对于它们的Sec对应物更高，这表明达特表达可以在一个步骤中产生相对纯的和高活性的蛋白质。
Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.
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影响因子：
3.2
作者：
Blaudeck, N;Sprenger, GA;Wiegert, T
通讯作者：
Wiegert, T
DOI：
10.1073/pnas.96.24.13703
发表时间：
1999-11-23
影响因子：
11.1
作者：
Bessette, PH;Åslund, F;Georgiou, G
通讯作者：
Georgiou, G
DOI：
10.1073/pnas.81.12.3830
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BENSON, SA;BREMER, E;SILHAVY, TJ
通讯作者：
SILHAVY, TJ
影响因子：
2.9
作者：
Dekker, N;Cox, RC;Egmond, MR
通讯作者：
Egmond, MR
影响因子：
3.2
作者：
DERMAN, AI;BECKWITH, J
通讯作者：
BECKWITH, J