Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag.
Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag.
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DOI：
10.1186/s12934-016-0534-3
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发表时间：
2016-08-05
影响因子：
6.4
通讯作者：
Lin Z
中科院分区：
文献类型：
--
作者：
Zhao Q;Xu W;Xing L;Lin Z
最近，多肽在治疗方面的应用变得很有吸引力。然而，通过重组和化学合成高效生产中大型多肽(30-100个氨基酸[AA])仍然具有挑战性。我们先前报道了β结构短肽ELK16(LELKKLELKLK)在大肠杆菌细胞中诱导活性酶聚集体的形成，并开发了一种简化的蛋白质表达和纯化方法。在该方法中，使用由内含素分子和ELK16组成的可切割自聚集标签(CSAT)从活性聚集体中释放出纯度合理的重组多肽。在这项工作中，我们将CSAT方法扩展到一系列具有重要治疗用途的中到大型多肽的通用表达和纯化方案，包括人胰高血糖素样肽1(31aa)、B型利钠肽(32aa)、Exendin 4(39aa)、趋化因子(C-C基序)配体5(也称为RANTES，66aa)、基质细胞衍生因子1α(67aa)、胰岛素样生长因子1(70aa)和瘦素(146aa)。在内含素介导的切割后，可溶性多肽直接释放到上清液中，而不溶性多肽可以通过反相高效液相色谱复性和纯化。此外，在目标多肽的上游添加了一个N-末端硫氧还蛋白标签，可以通过肠激酶切割将其去除，从而为目标多肽生成自然的N-末端。在实验室范围内，这些多肽的最终产率在0.1g/mg湿细胞重量之间，μg/mg湿重。本研究提供了一种快速有效的方法来表达和纯化难以通过化学合成或通过普通重组方法生产的多肽。它特别适合于大肽、可能被降解的多肽以及对宿主有毒性作用的多肽。它可以大大减少下游加工的成本和时间，因此在工业生产和实验室应用中都是有用的。本文的在线版本(doi：10.1186/s12934-0160534-3)包含补充材料，授权用户可以使用。
Peptides have recently become attractive for therapeutic applications. However, efficient production of medium- to large-sized peptides (30–100 amino acids [aa]) remains challenging both by recombinant and chemical synthesis. We previously reported the formation of active enzyme aggregates in Escherichia coli cells induced by the short β-structured peptide ELK16 (LELELKLKLELELKLK) and developed a streamlined protein expression and purification approach. In this approach, a cleavable self-aggregating tag (cSAT) consisting of an intein molecule and ELK16 was used to release the recombinant peptides with reasonable purity from active aggregates. In this work, we extended the cSAT approach to a generalized expression and purification solution for a set of medium- to large-sized peptides with important therapeutic uses, including human glucagon-like peptide 1 (31 aa), B-type natriuretic peptide (32 aa), exendin 4 (39 aa), chemokine (C–C motif) ligand 5 (also known as RANTES, 66 aa), stromal cell-derived factor 1α (67 aa), insulin-like growth factor 1 (70 aa), and leptin (146 aa). After intein-mediated cleavage, the soluble peptides were released directly into the supernatant while insoluble peptides could be refolded and purified by reverse phase high-performance liquid chromatography. Additionally, an N-terminal thioredoxin tag was added upstream of the target peptides, which can be removed by enterokinase cleavage, generating native N-terminus for target peptides. Final yields of the peptides ranged from 0.1 to 1.8 μg/mg wet cell weight at laboratory scale. The approach described in this study provides a fast and efficient route to express and purify peptides that are difficult or expensive to produce by chemical synthesis or by ordinary recombinant methods. It is particularly well suited for large peptides, peptides likely to be degraded, and peptides that have toxic effects on the host. It can greatly reduce the cost and time of downstream processing, and thus may be useful for both industrial manufacture and laboratory applications. The online version of this article (doi:10.1186/s12934-016-0534-3) contains supplementary material, which is available to authorized users.