Stolz J, Ludwig A, Sauer N
Friedrich-Alexander Universität Erlangen-Nürnberg, Lehrstuhl Botanik II, Erlangen, Germany.
FEBS Lett. 1998 Nov 27;440(1-2):213-7. doi: 10.1016/s0014-5793(98)01454-9.
Phage display is a powerful technique for identifying specific ligands to a given target. In this work random peptides derived from the biotin accepting domain of the Klebsiella pneumoniae oxaloacetate decarboxylase were displayed on bacteriophage lambda heads to determine the minimal sequence length that is necessary to effect biotinylation in vivo. Phages with a functional biotinylation domain were identified after affinity purification with immobilised avidin. All biotinylated phages isolated this way were found to have a sequence of 66 amino acids from the parental protein in common. This minimal biotinylation domain is fully functional as a biotin acceptor and more resistant to proteolytic attack compared to domains of larger size derived from the same protein. The data present the first example of a posttranslational protein modification analysed in a phage display system. Moreover, a biotin domain of reduced size and improved stability was identified, that should be superior to the larger parental protein as a tag to generate biotinylated fusion proteins.
噬菌体展示是一种用于识别给定靶标的特定配体的强大技术。在这项工作中，源自肺炎克雷伯氏菌草酰乙酸脱羧酶生物素接受结构域的随机肽被展示在λ噬菌体头部，以确定体内实现生物素化所需的最小序列长度。用固定化抗生物素蛋白进行亲和纯化后，鉴定出具有功能性生物素化结构域的噬菌体。发现以这种方式分离的所有生物素化噬菌体都具有来自亲本蛋白的66个氨基酸的共同序列。这个最小的生物素化结构域作为生物素受体具有完全功能，并且与来自同一蛋白的更大尺寸的结构域相比，对蛋白水解攻击更具抗性。这些数据展示了在噬菌体展示系统中分析的翻译后蛋白质修饰的首个实例。此外，还鉴定出了一个尺寸减小且稳定性提高了的生物素结构域，作为产生生物素化融合蛋白的标签，它应优于更大的亲本蛋白。
