Binding and enrichment of Escherichia coli spheroplasts expressing inner membrane tethered scFv antibodies on surface immobilized antigens
Binding and enrichment of Escherichia coli spheroplasts expressing inner membrane tethered scFv antibodies on surface immobilized antigens
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DOI：
10.1002/bit.21405
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发表时间：
2007-09
影响因子：
3.8
通讯作者：
S. Jung;K. Jeong;B. Iverson;G. Georgiou
中科院分区：
文献类型：
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作者：
S. Jung;K. Jeong;B. Iverson;G. Georgiou
锚定周质表达（APEx）是用于从大型组合文库中分离高亲和力配体结合蛋白的新方法（Harvey等人，2004，Proc Natl Acad Sci USA 101（25）：9193-9198）。在APEx中，蛋白质表达为与膜锚的融合物，所述膜锚将蛋白质束缚在大肠杆菌内膜的周质侧上。将细胞转化为原生质球并与可溶性荧光缀合配体孵育，导致表达配体结合蛋白的细胞的特异性标记，并随后通过流式细胞术分离。在这里，我们表明，在APEx格式表达的scFv抗体片段允许结合的原生质球固定的配体。在大肠杆菌中表达了特异于强心苷地高辛或炭疽杆菌保护性抗原（PA）的单链抗体作为阴性对照。大肠杆菌作为融合到N-末端或C-末端膜锚定结构域。只有C-末端锚定融合导致原生质球特异性识别和结合到具有固定抗原的TentaGel珠上。经过三轮流式细胞术筛选后，表达抗地高辛scFvs的原生质球从大量过量（1，000 ×）表达抗PA抗体的原生质球中富集了950倍。这些结果表明，APEx技术可用于基于与可能包括细胞表面上的抗原的不溶性抗原的结合来筛选文库。Biotechnol. Bioeng. 2007; 98：39-47.© 2007 Wiley Periodicals，Inc.
Anchored periplasmic expression (APEx) is a new method for the isolation of high affinity ligand‐binding proteins from large combinatorial libraries (Harvey et al., 2004, Proc Natl Acad Sci USA 101(25): 9193–9198). In APEx, proteins are expressed as fusions to a membrane anchor that tethers them onto the periplasmic side of the Escherichia coli inner membrane. Conversion of the cells into spheroplasts and incubation with soluble fluorescently conjugated ligands results in the specific labeling of cells expressing ligand‐binding proteins and their subsequent isolation by flow cytometry. Here we show that scFv antibody fragments expressed in the APEx format allow the binding of spheroplasts to immobilized ligands. ScFv antibodies specific for the cardiac glycoside digoxin or for the protective antigen (PA) of Bacillus anthracis as a negative control were expressed in E. coli as fusions to either N‐terminal or C‐terminal membrane anchoring domains. Only the C‐terminally anchored fusions resulted in specific recognition and binding of spheroplasts onto TentaGel beads with immobilized antigen. Following three rounds of flow cytometric screening, spheroplasts expressing anti‐digoxin scFvs were enriched 950‐fold from a large excess (1,000×) of spheroplasts expressing anti‐PA antibodies. These results indicate that the APEx technology may be employed for the screening of libraries based on binding to insoluble antigens possibly including antigens on cell surfaces. Biotechnol. Bioeng. 2007; 98: 39–47. © 2007 Wiley Periodicals, Inc.