Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
Protein Pept Lett. 2021;28(6):603-611. doi: 10.2174/0929866527666201223094249.
The unique hypervariable C-terminal region (HVR) of K-Ras4B, one of the most frequently mutated proteins in many powerful cancers, contains a C-terminal farnesylated and methylated Cys and a poly-lysine motif, which decides the association of K-Ras4B to the inner leaflet of plasma membrane for activating the downstream signaling activity. In our previous work, we inserted an additional Cys in K-Ras4B HVR peptide synthesis for NCL in the semi-synthesis of K-Ras4b protein, but it is not suitable for application in protein dimerization research. The recently developed selenocysteine (Sec, U) mediated native chemical ligation reaction followed by selective deselenization, which can help to broaden the scope of protein synthesis, requires the generation of the peptide fragment with an N-terminal Sec.
To synthesize K-Ras4B HVR peptide containing both N-terminal Sec and C-terminal farnesylated and methylated Cys to achieve traceless protein semi-synthesis.
We have developed a facile synthesis approach for producing Boc-Sec)2-OH using economic Se powder, which can facilitate scaling up preparation of peptides containing Sec at the N-terminus. Furthermore, we synthesized K-Ras4B HVR peptide containing selenocystine by utilization of Boc-Sec)2-OH. Finally, we took K-Ras4B HVR peptide as an example to test the compatibility of farnesylation reaction at Cys with the N-terminal Sec)2, and the farnesyl group was successfully added to the thiol group of Cys.
K-Ras4B 是许多恶性肿瘤中突变最频繁的蛋白质之一，其独特的高变区（HVR）的 C 末端含有一个 C 末端法尼基化和甲基化的半胱氨酸和一个多赖氨酸基序，这决定了 K-Ras4B 与质膜内层的结合，从而激活下游信号转导活性。在我们之前的工作中，我们在 K-Ras4B HVR 肽合成中插入了一个额外的半胱氨酸，用于 K-Ras4b 蛋白的半合成中的非天然 C 端连接（NCL），但它不适合用于蛋白质二聚化研究。最近开发的硒代半胱氨酸（Sec，U）介导的天然化学连接反应随后进行选择性去硒化，可以帮助拓宽蛋白质合成的范围，需要生成具有 N 端 Sec 的肽片段。
合成同时含有 N 端 Sec 和 C 末端法尼基化和甲基化半胱氨酸的 K-Ras4B HVR 肽，以实现无痕蛋白质半合成。
我们开发了一种简便的合成 Boc-Sec)2-OH 的方法，使用经济的 Se 粉，这可以促进含 Sec 的 N 端肽的大规模制备。此外，我们利用 Boc-Sec)2-OH 合成了含有硒代半胱氨酸的 K-Ras4B HVR 肽。最后，我们以 K-Ras4B HVR 肽为例，测试了 Cys 上的法尼基化反应与 N 端 Sec)2 的兼容性，法尼基基团成功地添加到了 Cys 的巯基上。
