Maenaka K, Furuta M, Tsumoto K, Watanabe K, Ueda Y, Kumagai I
Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
Biochem Biophys Res Commun. 1996 Jan 26;218(3):682-7. doi: 10.1006/bbrc.1996.0122.
A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed. A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII fusion protein in the opposite direction to that of the lac promoter of pTZ18U. Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E. coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL10, could be stably and functionally displayed. Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII. Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions.
构建了一种用于在丝状噬菌体表面展示pIII融合蛋白的稳定表达系统。通过将编码pIII融合蛋白的基因以与pTZ18U的lac启动子相反的方向插入，构建了一个噬菌粒pIII展示载体pLUCK。利用该噬菌体展示系统，两种酶，即鸡蛋清溶菌酶（HEL）和大肠杆菌碱性磷酸酶，以及抗HEL单克隆抗体HyHEL10的单链Fv片段，能够被稳定且功能性地展示。Northern印迹和引物延伸分析表明，编码pIII融合HEL的少量正义mRNA是从位于编码pIII C末端区域的次要噬菌体启动子转录而来的。pIII融合蛋白的抑制表达能够在丝状噬菌体上展示多种蛋白质，而无需严格的表达条件。
