High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.
High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.
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DOI：
10.1093/protein/gzh044
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发表时间：
2004-04
期刊：
影响因子：
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通讯作者：
T. Tenno;Natsuko Goda;Y. Tateishi;H. Tochio;M. Mishima;H. Hayashi;M. Shirakawa;H. Hiroaki
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文献类型：
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作者：
T. Tenno;Natsuko Goda;Y. Tateishi;H. Tochio;M. Mishima;H. Hayashi;M. Shirakawa;H. Hiroaki
用114个氨基酸的硫氧还蛋白（TRX）产生标记肽的融合蛋白构建体，结合组氨酸标签用于亲和纯化。根据我们的新的单向TA克隆方法PRESAT-载体，在融合载体的多克隆位点设计了两个串联的AhdI位点，允许一步无背景克隆DNA片段。构建体被设计为掺入四个残基序列Ile-Asp-Gly-Arg，以在融合蛋白的Xa因子切割后产生纯肽。该系统是有效的和成本效益的同位素标记的肽异构NMR研究。七个不同长度的肽，包括垂体腺苷酸环化酶激活多肽（PACAP），血管活性肠肽（VIP）和泛素相互作用基序（UIM），表达使用该TRX融合系统在所有情况下得到可溶性融合蛋白的构建体。根据肽的长度，应用三种替代方法制备DNA片段，例如聚合酶链反应、化学合成或“半合成方法”，其是化学合成和酶促延伸的组合。以高通量方式容易地构建、表达和纯化重组肽的能力将在生物医学研究和药物发现领域中具有巨大的益处。
Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.