Semisynthesis of cytotoxic proteins using a modified protein splicing element
Semisynthesis of cytotoxic proteins using a modified protein splicing element
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DOI：
10.1002/pro.5560071103
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发表时间：
1998-11-01
期刊：
影响因子：
8
通讯作者：
Xu, MQ
中科院分区：
文献类型：
--
作者：
Evans, TC;Benner, J;Xu, MQ
两种细胞毒性蛋白，即牛胰腺核糖核酸酶 A (RNase A) 和来自副流感嗜血杆菌的限制性内切核酸酶 (HpaI)，是使用一种新型半合成方法生产的，该方法利用蛋白质剪接元件（内含肽）在重组蛋白的 C 末端生成反应性硫酯。合成肽的 N 端半胱氨酸对该硫酯的亲核攻击最终导致两种反应物通过天然肽键连接。该策略用于通过分离这些蛋白质的无活性截短形式（分别是 RNase A 和 HpaI 的前 109 和 223 个氨基酸）作为由靶蛋白、内含肽和几丁质结合域组成的融合蛋白来生产 RNase A 和 HpaI。硫醇诱导的前体裂解导致释放带有 C 末端硫酯标签的靶蛋白。添加代表截短形式中缺失的氨基酸的合成肽导致产生全长产物，其显示出指示野生型酶的催化活性。连接和复性 RNase A 的周转数和 K-m 分别为 8.2 s(-1) 和 1.5 mM，与报道的 8.3 s(-1) 和 1.2 mM 值非常一致（Hodges & Menifield，1975）。连接的 HpaI 的比活性为 0.5-1.5 x 10(6) U/mg，与 1-2 x 10(6) U/mg 的预期值相比毫不逊色（J. Benner，未发表的观察结果）。除了协助细胞毒性蛋白的产生外，该技术还可以轻松地将非天然氨基酸插入到蛋白质序列中。
Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and K-m, for ligated and renatured RNase A were 8.2 s(-1) and 1.5 mM, in good agreement with reported values of 8.3 s(-1) and 1.2 mM (Hodges & Menifield, 1975). Ligated HpaI had a specific activity of 0.5-1.5 x 10(6) U/mg, which compared favorably with the expected value of 1-2 x 10(6) U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.