Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies.
Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies.
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DOI：
10.1371/journal.pone.0075126
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Fernández LÁ
中科院分区：
文献类型：
--
作者：
Salema V;Marín E;Martínez-Arteaga R;Ruano-Gallego D;Fraile S;Margolles Y;Teira X;Gutierrez C;Bodelón G;Fernández LÁ
由于外膜(OM)的存在，直接展示在大肠杆菌细胞表面的抗体(Ab)文库的筛选受到阻碍。在这项工作中，我们证明了EhaA自身转运蛋白和内膜蛋白的天然β结构域，这两个在OM中具有相反拓扑结构的肠出血性大肠杆菌O157：H7蛋白，是在大肠杆菌K-12细胞表面展示来自骆驼胶体(纳米体或VHH)的单域抗体(SdAbs)的有效系统，并用于利用磁性细胞分选(MACs)筛选高亲和力sdAbb。我们分析了EhaA和内膜蛋白β结构域展示单个单抗和单抗文库的能力，这些单抗和单抗文库是用来自EHEC的移位内膜受体胞外区免疫后获得的。我们证明，这两个系统都在大肠杆菌细胞表面展示了功能性的sdAb，蛋白降解和细胞毒性很小，尽管展示了带有内膜β结构域的sdAb的大肠杆菌细胞表现出更高的抗原结合能力。用MACS对两个大肠杆菌展示文库进行TirMEHEC结合克隆筛选。两种展示系统都选择了高亲和力的结合体，尽管内蛋白β结构域的结合效率更高。经细胞流式细胞术、酶联免疫吸附试验和纯化的sdAbs表面等离子共振检测，证明了所选克隆具有抗TirMEHEC的特异性。最后，我们利用大肠杆菌细胞展示系统在平衡条件下通过流式细胞术分析对所选sdAb的亲和力进行了估计。
Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.
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影响因子：
3.6
作者：
BLOMFIELD, IC;MCCLAIN, MS;EISENSTEIN, BI
通讯作者：
EISENSTEIN, BI
DOI：
10.1093/protein/11.9.825
发表时间：
1998-09-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Daugherty, PS;Chen, G;Georgiou, G
通讯作者：
Georgiou, G
影响因子：
100.3
作者：
Beck, Alain;Wurch, Thierry;Corvaia, Nathalie
通讯作者：
Corvaia, Nathalie
DOI：
10.1093/protein/9.2.239
发表时间：
1996-02-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Georgiou, G;Stephens, DL;Earhart, CF
通讯作者：
Earhart, CF
影响因子：
2.4
作者：
Holt, Lucy J.;Basran, Amrik;Tomlinson, Ian M.
通讯作者：
Tomlinson, Ian M.