Berdichevsky Y, Ben-Zeev E, Lamed R, Benhar I
Department of Molecular Microbiology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Tel-Aviv University, Ramat Aviv 69978, Israel.
J Immunol Methods. 1999 Aug 31;228(1-2):151-62. doi: 10.1016/s0022-1759(99)00096-4.
Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vitro evolution of these biologically important molecules. However, the general usefulness of this technology is still limited by some technical difficulties. One of the most debilitating obstacles to the widespread application of the technology is the accumulation of "insert loss" clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted. Another difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned. Here, we report the construction of a novel phage-display system designed to address these problems. In our system a single-chain Fv (scFv) is expressed as an in-frame fusion protein with a cellulose-binding domain (CBD) derived from the Clostridium thermocellum cellulosome. The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof. We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires.
抗体片段的噬菌体展示已被证明是用于分离和体外进化这些具有重要生物学意义分子的强大工具。然而，这项技术的普遍实用性仍受到一些技术难题的限制。该技术广泛应用的最棘手障碍之一是文库中“插入缺失”克隆的积累；即编码部分或全部克隆抗体片段的DNA已被删除的噬菌粒克隆。当噬菌体技术用于克隆杂交瘤衍生的抗体基因时会出现另一个难题，其中可能会偶然克隆到与杂交瘤抗体特异性无关的骨髓瘤衍生轻链。在此，我们报告构建了一种旨在解决这些问题的新型噬菌体展示系统。在我们的系统中，单链Fv（scFv）作为与源自嗜热栖热菌纤维小体的纤维素结合结构域（CBD）的框内融合蛋白表达。CBD结构域用作亲和标签，可从粗培养上清液中快速捕获和浓缩噬菌体，并对展示噬菌体及其产生的可溶性scFv进行免疫检测。我们证明了我们的系统在解决上述技术难题以及加速从组合抗体库中分离scFv过程中的实用性。
