Zwick M B, Bonnycastle L L, Noren K A, Venturini S, Leong E, Barbas C F, Noren C J, Scott J K
Biochemistry Program, Department of Biological Sciences, Institute of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada.
Anal Biochem. 1998 Nov 1;264(1):87-97. doi: 10.1006/abio.1998.2793.
Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII- and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.
随机肽库以与次要外壳蛋白pIII或主要外壳蛋白pVIII融合的形式展示在丝状噬菌体上。我们设计了一种方法，通过将展示在噬菌体克隆上的肽转移到由malE编码的大肠杆菌麦芽糖结合蛋白（MBP）的N端，来分离该肽。将肽序列转移到单体MBP上可消除插入肽下游的噬菌体编码氨基酸以及噬菌体上多价展示所引起的亲和力效应。肽:MBP融合蛋白也很容易在直链淀粉柱上进行亲和纯化。对pMal-p2载体进行了改造，使其能够接受编码与各自前导序列融合的pIII和pVIII展示肽的噬菌体DNA。两种类型的前导序列均显示可将肽:MBP融合蛋白靶向大肠杆菌的周质。通过用HIV-1特异性单克隆抗体（Ab）筛选，将一种将肽转移到MBP的简化程序应用于从一组pVIII展示肽库中分离出的克隆。通过酶联免疫吸附测定法，该抗体与每种肽:MBP融合蛋白结合，并且每种肽中都需要存在二硫键。还使用表面等离子体共振对一些肽:MBP融合蛋白进行了分析。因此，我们的研究表明了malE融合载体在表征噬菌体展示肽方面的价值。
