School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
Microb Biotechnol. 2008 Sep;1(5):403-15. doi: 10.1111/j.1751-7915.2008.00041.x.
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.
从历史上看，革兰氏阴性细菌的一般分泌（Sec）途径一直是将异源蛋白递送至许多表达和工程应用中外周质的主要途径。在这里，我们系统地研究了双精氨酸转运（Tat）途径作为异源蛋白的替代途径，可能具有优势。总的来说，我们发现：（i）模型底物的出口效率和周质产量受 Tat 信号肽组成的影响，（ii）Tat 底物在到达周质时在其 N 末端正确加工，（iii）与麦芽糖结合蛋白（MBP）融合的蛋白可通过 Tat 系统可靠地输出，但只有在正确折叠的情况下；异常折叠的 MBP 融合体被 Tat 途径的折叠质量控制特性排除。我们还观察到，对于相对较小、折叠良好的蛋白质，Tat 出口产量与 Sec 相当，对于需要细胞质折叠的蛋白质，Tat 相对于 Sec 更高，对于较大的可溶性融合蛋白，Tat 相对于 Sec 更低。有趣的是，与 Sec 对应物相比，某些 Tat 底物从周质中纯化得到的物质的比活性更高，这表明 Tat 表达可以一步产生相对纯净和高活性的蛋白质。
