PRODUCTION AND FLUORESCENCE-ACTIVATED CELL SORTING OF ESCHERICHIA-COLI EXPRESSING A FUNCTIONAL ANTIBODY FRAGMENT ON THE EXTERNAL SURFACE
PRODUCTION AND FLUORESCENCE-ACTIVATED CELL SORTING OF ESCHERICHIA-COLI EXPRESSING A FUNCTIONAL ANTIBODY FRAGMENT ON THE EXTERNAL SURFACE
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DOI：
10.1073/pnas.90.22.10444
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发表时间：
1993-11-15
影响因子：
11.1
通讯作者：
GEORGIOU, G
中科院分区：
文献类型：
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作者：
FRANCISCO, JA;CAMPBELL, R;GEORGIOU, G
我们在大肠杆菌的外表面上表达了一个单链 Fv (scFv) 抗体片段，该片段由来自两个独立的抗地高辛单克隆抗体的可变重结构域和可变轻结构域组成，方法是将其与先前显示的 Lpp-OmpA 杂交体融合，将异源蛋白引导至细胞表面。这种 scFv 融合体以高水平表达，并显示出以高亲和力和特异性结合半抗原。全细胞 ELISA、荧光显微镜、蛋白酶敏感性和流式细胞术均证实 scFv 锚定在外膜上并且可在表面上接触。利用荧光激活细胞分选，我们只需两步即可从 10(5) 倍过量的对照细胞中特异性富集 scFv 产生细胞。大肠杆菌表面上抗体片段的表达正在被评估为体外生产和选择有用抗体片段的一种有吸引力的方法。
We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.