pFab60: A new, efficient vector for expression of antibody Fab fragments displayed on phage
pFab60: A new, efficient vector for expression of antibody Fab fragments displayed on phage
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DOI：
10.1093/protein/8.10.1063
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发表时间：
1995-10-01
期刊：
影响因子：
--
通讯作者：
Engberg, J
中科院分区：
文献类型：
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作者：
Johansen, LK;Albrechtsen, B;Engberg, J
结合噬菌体展示、抗体基因聚合酶链反应（PCR）扩增和大肠杆菌高效表达功能性抗体片段，可以建立大型抗体片段展示文库，快速分离特异性抗体片段（Huse et al., 1989; Barbas et al., 1991; Clackson et al., 1991）。编码抗体片段的重链和轻链的基因被克隆到一个噬菌体中，其中一条链与丝状噬菌体的次要外壳蛋白“药丸”融合表达。在质周空间内，药丸被固定在膜上，重链和轻链组装形成功能性抗体片段。在被辅助噬菌体感染后，一些野生型pin被抗体片段pin融合蛋白取代，抗体片段显示在噬菌体的尖端，这使得结合抗原的噬菌体可以选择性富集。在这里，我们提出了一种噬菌体载体pFab60，设计用于分离克隆整个Kappa基因和VH（或Fd）小鼠基因片段，相对于其他人发表的研究成果（Barbas et al., 1991; Soderlind et al., 1993），该载体有若干改进。它(i)从单个双电子P/ac驱动的表达盒中表达轻、重Fd链，从而避免了两个相同启动子区域之间的同源重组；（ii）表达lac抑制因子，使其保持紧密抑制，不受菌株背景的影响；（iii）表达截断版的pill （Apm）。（iv）包含胰蛋白酶裂解位点的序列，该序列允许洗脱与抗原结合的Fab噬菌体独立于其抗原结合强度；(v)包含下游（His） 6标签序列，该序列在去除Agin后极大地促进了所得可溶性Fab分子的纯化，而不依赖于其抗原结合特性。利用该载体，我们构建了共有bb10108个成员的小鼠文库，表明胰蛋白酶在生物筛选过程中是一种温和高效的Fab噬菌体洗脱剂，并且我们在固定化金属亲和层析（IMAC）柱上一步纯化了（His） 6标记的抗体Fab片段。最后，我们在不同的宿主菌株中表达了Fab片段，发现不同菌株在噬菌体上产生功能性Fab片段的能力存在显著差异。
By combining phage display, polymerase chain reaction (PCR) amplification of antibody genes, and Escherichia coli's efficient expression of functional antibody fragments, it is possible to make large display libraries of antibody fragments and to isolate specific antibody fragments rapidly (Huse et al., 1989; Barbas et al., 1991; Clackson et al., 1991). The genes that encode the heavy and light chains of antibody fragments are cloned into a phagemid such that one chain is expressed in fusion with the minor coat protein, pill, of a filamentous bacteriophage. In the periplasmic space, pill is anchored to the membrane and the heavy and light chain assemble to form a functional antibody fragment. Upon infection with helper phage, some wild type pin are displaced by antibody fragmentpin fusion protein, and the antibody fragment is displayed on the tip of the phage, which allows for selective enrichment of phages that bind antigen.Here we present a phagemid vector, pFab60, designed for separate cloning of the entire Kappa gene and the VH (or Fd) murine gene fragments, which represents several improvements relative to those published by others (Barbas et al., 1991; Soderlind et al., 1993). It (i) expresses the light and heavy Fd chains from a single bicistronic P/ac-driven expression cassette, thereby avoiding homologous recombination between two identical promoter regions,(ii) expresses lac repressor which keeps the cassette tightly repressed independently of strain background,(iii) expresses a truncated version of pill (Apm), thereby reducing immunity to superinfection caused by intact pill,(iv) contains the sequence for a trypsin cleavage site which permits elution of antigen-bound Fab phages independent of their antigen-binding strength and (v) contains a downstream (His) 6 tag sequence, which upon removal of Agin greatly facilitates purification of the resulting soluble Fab molecules independent of their antigen-binding properties. Using this vector, we have constructed murine libraries with a total of> 108 members, shown that trypsin works as a gentle and efficient Fab phage elutor in bio-panning procedures, and we have purified (His) 6-tagged antibody Fab fragments in one step on immobilized metal affinity chromatography (IMAC) columns. Finally, we have expressed Fab fragments in different host strains and found a significant difference in the various strains' abilities to produce functional Fab fragments on phage.