Department of Systems Biochemistry, Faculty of Medicine, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany.
Methods Mol Biol. 2023;2643:383-390. doi: 10.1007/978-1-0716-3048-8_27.
The translation of mRNAs lacking a stop codon results in a nascent polypeptide chain still attached to the translating ribosome. When containing an exposed N-terminal targeting signal, these so-called nonstop (ns) proteins have been shown to localize to their respective organellar translocation channel, resulting in stabilized translocation intermediates. Utilizing a plasmid encoding a FLAG-tagged nonstop protein with an N-terminal targeting signal early-stage ribosome-associated protein complexes can be purified by affinity chromatography. This will be exemplified by purification of protein complexes of the peroxisomal protein import machinery using different nonstop variants of the PTS2 cargo protein Fox3p from both soluble and membrane fractions.
无终止密码子的 mRNA 的翻译导致新生多肽链仍然与翻译核糖体相连。当含有暴露的 N 端靶向信号时，这些所谓的无终止（ns）蛋白已被证明定位于其各自的细胞器易位通道，导致稳定的易位中间体。利用编码具有 N 端靶向信号的 FLAG 标记无终止蛋白的质粒，可以通过亲和层析纯化早期核糖体相关蛋白复合物。这将通过使用来自可溶性和膜部分的 PTS2 货物蛋白 Fox3p 的不同无终止变体从过氧化物酶体蛋白导入机制中纯化蛋白复合物来举例说明。
