Biosynthesis of the cyclotide Kalata B1 by using protein splicing
Biosynthesis of the cyclotide Kalata B1 by using protein splicing
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DOI：
10.1002/anie.200503882
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Camarero, JA
中科院分区：
文献类型：
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作者：
Kimura, RH;Tran, AT;Camarero, JA
环肽中的核心结构基序被称为环状胱氨酸结（CCK），其特征是胱氨酸结嵌入圆形主链拓扑中。[1d] 胱氨酸结涉及两个二硫键，形成一个环，第三个二硫键穿透该环。独特的环状主链拓扑和三个二硫键的打结排列赋予环肽优异的稳定性以及对化学、酶和热降解的抵抗力。 [2]此外，它们明确的结构与一系列生物功能相关，例如子宫收缩活性、抑制胰蛋白酶和神经紧张素结合、细胞毒性、抗艾滋病毒、抗菌和杀虫活性。[1b-c]总而言之，这些特征表明环肽是开发稳定肽药物的理想分子支架。[1b]尽管环状肽的化学合成已经得到很好的探索，并且存在许多涉及固相或液相的不同方法，[3]最近分子生物学和蛋白质工程领域的发展现已使环肽的生物合成成为可能。这一进展主要在两个领域取得，即非核糖体肽合成[4]和表达蛋白连接（EPL）/蛋白转拼。[5]通过使用重组DNA表达技术获得生物合成环肽提供了产生高度稳定的环状多肽的大型组合文库的令人兴奋的可能性。 This would allow the generation of cell-based combinatorial libraries that could be screened either in vitro or in vivo for their ability to regulate cellular processes.在此，我们描述了使用工程化内含肽生物合成环肽 KalataB1 (KB1)。我们的方法（图 1）基于天然化学连接 (NCL) 的分子内版本。 [6] NCL 涉及一种肽的 N 末端 Cys 残基与另一种肽的 α-硫酯基团之间的化学选择性反应。重要的是，将这两个基团掺入同一合成多肽中会导致有效的环化。 [3]为了测试这种方法，我们构建了几个编码不同 KB1 线性前体的质粒（图 2）和
The core structural motif in cyclotides has been termed a cyclic cystine knot (CCK) and is characterized by a cystine knot that is embedded into a circular backbone topology.[1d] The cystine knot involves two disulfide bonds that form a ring that is penetrated by a third disulfide bond. The unique cyclicbackbone topology and knotted arrangement of the three disulfide bonds endow the cyclotides with exceptional stability and resistance to chemical, enzymatic, and thermal degradation.[2] Furthermore, their well-defined structures have been associated with a range of biological functions such as uterotonic activity, inhibition of trypsin and neurotension binding, cytotoxicity, anti-HIV, antimicrobial, and insecticidal activity.[1b–c] Together, these characteristics suggest that cyclotides are ideal molecular scaffolds for the development of stable peptide drugs.[1b] Despite the fact that the chemical synthesis of circular peptides has been well explored and a number of different approaches involving the solid or liquid phases exist,[3] recent developments in the fields of molecular biology and protein engineering have now made possible the biosynthesis of cyclic peptides. This progress has been made mainly in two areas, nonribosomal peptide synthesis [4] and expressed protein ligation (EPL)/protein trans-splicing.[5] Access to biosynthetic cyclotides by using techniques of recombinant-DNA expression offers the exciting possibility of producing large combinatorial libraries of highly stable cyclic polypeptides. This would allow the generation of cell-based combinatorial libraries that could be screened either in vitro or in vivo for their ability to regulate cellular processes. Herein, we describe the biosynthesis of the cyclotide KalataB1 (KB1) by using an engineered intein. Our approach (Figure 1) is based on an intramolecular version of native chemical ligation (NCL).[6] NCL involves the chemoselective reaction between an N-terminal Cys residue of one peptide and an α-thioester group of a second peptide. Importantly, incorporation of these two groups into the same synthetic polypeptide leads to efficient circularization.[3] To test this approach, we constructed several plasmids that encode different KB1 linear precursors (Figure 2) and