MacBeath G, Kast P
Scripps Research Institute, La Jolla, CA, USA.
Biotechniques. 1998 May;24(5):789-94. doi: 10.2144/98245st02.
pET and similar vectors are widely used for efficient gene expression in Escherichia coli and subsequent protein purification, often by means of a C-terminal histidine (His) tag. We found that the TGA translation termination signal following the His-tag sequence in pET constructs gives rise to a significant fraction of read-through protein extended by 21 amino acids. Mass spectrometry indicated that tryptophan is inserted at the UGA (opal) stop codon in the examined non-opal suppressor strains; no evidence for translational frameshifting was detected. We have shown that the problem of obtaining heterogeneous protein preparations can easily be corrected. Plasmid pATCH1 provides a replacement sequence for the inefficient stop signal and can be used to repair both pET vectors and existing pET-based expression constructs. Our observation illustrates the largely ignored fact that a UGA codon is the worst choice for proper translation termination in efficient overexpression vectors.
pET及类似载体广泛用于在大肠杆菌中高效表达基因及后续蛋白质纯化，通常借助C端组氨酸（His）标签。我们发现，pET构建体中His标签序列后的TGA翻译终止信号会导致相当一部分通读蛋白延长21个氨基酸。质谱分析表明，在所检测的非琥珀抑制菌株中，色氨酸插入到UGA（乳白）终止密码子处；未检测到翻译移码的证据。我们已经表明，获得异质蛋白质制剂的问题很容易得到纠正。质粒pATCH1为低效终止信号提供了一个替代序列，可用于修复pET载体和现有的基于pET的表达构建体。我们的观察结果说明了一个很大程度上被忽视的事实，即在高效过表达载体中，UGA密码子是正确翻译终止的最糟糕选择。
