Huovinen Tuomas, Syrjänpää Markku, Sanmark Hanna, Seppä Titta, Akter Sultana, Khan Liton Md Ferdhos, Lamminmäki Urpo
Department of Biochemistry, University of Turku, Turku 20520, Finland.
BMC Res Notes. 2014 Sep 19;7:661. doi: 10.1186/1756-0500-7-661.
Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins.
In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection.
The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.
丝状噬菌体展示已成为工程化抗体片段的常用工具。几种衣壳蛋白已被用于展示抗体，其中基因III（p3）蛋白应用最为广泛，其次是基因IX（p9）蛋白的相关实验。尽管广受欢迎，但尚无文库规模的研究来客观比较通过不同衣壳蛋白展示时文库筛选性能的差异。
在本研究中，相同的抗体库以Fab片段形式展示在p9、p3和截短的p3（p3Δ）上。此外，文库克隆以单链抗体片段（ScFv）形式展示在p3Δ上，并且通过超噬菌体和VCS-M13辅助噬菌体感染调节Fab-p3展示的价态。在针对链霉亲和素（STR）和地高辛（DIG）的重复平行淘选反应中跟踪文库的筛选性能。所有展示形式的筛选均成功，但从Fab-p9文库中富集特异性克隆的效率明显低于其他文库。从p3Δ展示中获得的输出多样性最高，从ScFv库中获得的抗DIG抗体亲和力最高。不幸的是，检索到的特异性克隆数量过低，无法明确分析每个文库中获得的独特克隆数量的差异。然而，在淘选之前，p3-Fab文库中的序列多样性严重降低，这反过来表现为独特特异性克隆数量较少。超噬菌体的多价展示导致独特克隆数量增加，但通过VCS-M13辅助噬菌体感染也回收了相同的最高亲和力抗DIG Fab。
在我们的实验中，作为与p9衣壳蛋白融合的Fab库中目标特异性克隆的富集受损，单噬菌体包装周期后Fab-p3文库中功能多样性的显著丧失，以及从相同来源库中作为ScFv分子比作为Fab分子检索到更高亲和力的抗地高辛克隆，表明所选的展示形式可能对筛选结果有重大影响。本研究表明，在规划定向进化实验时，除了文库内容外，还应考虑与展示相关的问题。
