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Systematic quality control in construction of phage surface Fab display library with a diversity of 10e11
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  • Kai Cui,
  • Yujun Yang,
  • Xiaoju Zhang,
  • Chen Zhou
Kai Cui
Zhengzhou University

Corresponding Author:[email protected]

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Yujun Yang
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Xiaoju Zhang
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Abstract

Phage display is widely used in the discovery and development of antibody therapeutics. Though the library quality is critical, no quality control system has been developed. Here, we report a systematic quality control system to ensure the fast construction of phage display libraries. The keys of the system include the single PCR amplification of variable heavy (VH) and light chain (LC) repertoires, construction of component libraries, introduction of a linker between the LC and VH regions in display vectors and use of four-fragments ligation (four-way ligation). Using this system, two Fab display libraries (4 × 1010 and 1.6 × 1011) were constructed. Titration, sequencing and expression analyses of the libraries revealed a ligation-transformation efficiency (LTE) of >109 clones/µg of ligation product, >80% unique clones and >80% Fab-expressible clones, indicating a diversity of > 1.28 x 1011. Starting from the amplification of antibody gene repertoires, a library up to 1011 can be constructed in three months. The results demonstrated the practicability, simplicity, reliability, scalability, flexibility, and reproducibility of the system in the fast construction of a high-quality phage display library.