Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

The key for a successful isolation of antibodies is the phage library used for the selection [30] . Various methods have been employed to make antibody phage display libraries. Most widely used methods consist of PCR amplification of antibody fragments, followed by enzymatic digestion and ligation with the vector. These methods are time consuming and have a low efficiency in the ligation and transformation step. In the present study, we developed the EASeL method to construct a large shark V NAR phage library. We used a labeling PCR to add homologue regions to the V NAR pool. Then an overlap extension PCR was used to assemble V NAR and phagemid DNA, followed by self-ligation of the whole library DNA. It only took several weeks to make the shark V NAR library while conventional approaches would require several months. Our protocol has significantly shortened the time required to construct a large gene library. Another contemporary method used for gene cloning is "Gibson Assembly". This method is based on enzymatic assembly for joining multiple overlapping DNA fragments into a single reaction system [31] . Our method may be more efficient than Gibson Assembly. Gibson Assembly requires 5-exonuclease to nick the 5-terminals of the DNA fragments to make them complementary followed by annealing together. DNA polymerase was then used to fill the gap, and DNA ligase was used to seal the gap. Therefore, the two termini that Antibody Therapeutics, 2019 7 are being ligated have to be identical for each other for ∼20 nucleotides. The Gibson Assembly reaction may fail due to complications related to total repeat density (direct, inverse, and palindromic repeat elements), extremes in G/C content, and secondary structures near the 3' and 5' termini of the sequence. To our best knowledge, there is no report using the Gibson Assembly for the development of a large phage-displayed antibody library. In our method, we only need the conventional T4 DNA ligase to run a standard ligation at 16 • C for 12-24 h. The two ends of the DNA are blunt and do not need to be identical for blunt-end ligation. Therefore, our method is similar to traditional blunt-end ligation, and the terminal part of the DNA fragment sequence can be varied. During the preparation of this manuscript, our laboratory has applied the EASeL method to successfully make 20 large camel V H H singledomain phage libraries. Taken together, the new EASeL library construction method is robust, comprehensive, and quick.