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 V NAR domain is an Ig superfamily domain with two β sheets held together by two canonical cysteine residues in framework regions (FRs) 1 and 3. In addition to these canonical cysteines, complementarity determining region 3 (CDR3) can have one or two additional cysteines forming additional disulfide bonds within the V domain. IgNARs are classified into four types based on the number and position of non-canonical cysteines in the V NAR domain [14] . Type I V NAR contains two non-canonical cysteine residues in CDR3 encoded by the diversity region or by N-nucleotide additions that form two disulfide bonds with FR2 and 4. Interestingly, Type I has only been reported in nurse sharks, Ginglymostoma cirratum [14] . Type II V NAR domains form disulfide bonds between one Dencoded non-canonical cysteine in CDR3 and another non-canonical cysteine in CDR1. Type III is similar to Type II except there is a highly conserved tryptophan residue in CDR1 positioned adjacent to the disulfide bond. Type IV has no non-canonical disulfide bonds as found in other three V NAR types. Both Type I and Type II V NAR s have protruding CDR3s that enable binding to pockets and grooves [15, 16] . Classical IgG and camelid V H H contain a CDR2 loop that is not present in shark V NAR s and are replaced with highly diverse amino acids, termed hypervariable region 2 (HV2) [11] . Additionally, there is a second hypervariable region, named HV4, which is inserted in the middle of FR3, therefore separating FR3 into FR3a and FR3b.