Protein chemical synthesis by α-ketoacid-hydroxylamine ligation
Protein chemical synthesis by α-ketoacid-hydroxylamine ligation
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DOI：
10.1038/nprot.2016.052
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发表时间：
2016-06-01
期刊：
影响因子：
14.8
通讯作者：
Bode, Jeffrey W.
中科院分区：
文献类型：
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作者：
Harmand, Thibault J.;Murar, Claudia E.;Bode, Jeffrey W.
蛋白质的全化学合成使研究人员能够定制设计蛋白质，而无需插入非天然氨基所需的复杂分子生物学或依赖于细胞中过表达的方法所产生的生物污染。我们描述了一个详细的程序与（γ-酮酸-羟胺（KAHA连接）的蛋白质的化学合成，使用（S）-5-oxaprotine（Opr）作为一个关键的构建块。该方案包括两个主要部分：（i）通过标准芴基甲氧羰基（Fmoc）化学合成肽片段和（ii）含有Opr和C-末端肽ts-酮酸的片段之间的KAHA连接。该程序提供了天然化学连接（NCL）的替代方案，其对于蛋白质的合成可能是有价值的，特别是不含半胱氨酸残基的靶标。连接条件-酸性DMSO/H2O或N-甲基-2-吡咯烷酮（NMP）/H2O-理想地适合于溶解肽段，包括许多疏水性实例。该方案的实用性和效率通过成熟betatrophin（也称为ANGPTL 8）的全化学合成来证明，所述成熟betatrophin是一种不含半胱氨酸残基的177个残基的蛋白质。通过该方案，betatrophin蛋白的全合成在约35个工作日内以数毫克规模实现。
Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino adds or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the (y-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaprotine (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide ts-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.