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. 2008 Apr 11;74(11):3497–3511. doi: 10.1128/AEM.02844-07

FIG. 2.

FIG. 2.

Crybody libraries and their construction. (a) Schematic representation of the seven Crybody libraries derived from cry1Aa13 and a human scFv antibody library. Sequences encoding loop 1, loop 2, and loop 3 of cry1Aa13 are shown in blue, green, and yellow, respectively. CDR-H3 sequences are shown in red. The three domains encoded by cry1Aa13 are labeled I, II, and III. VL and VH indicate the antibody domains. (b) Sequences of Crybodies with CDR-H3 insertions at site 1, 2, or 3. Adaptor codons are shown in bold, and the general CDR-H3 sequence is in red. The block arrows show secondary structure as defined by Grochulski et al. (30). (c) Overview of the three-step assembly strategy used to construct the cc123 library, involving (1) the recovery of CDR-H3 sequences from the scFv antibody library and their ligation to synthetic double-stranded DNA adaptor molecules corresponding to sequences adjacent to cry1Aa13 domain II loops, (2) PCR amplification of cry1Aa13 sequences encoding the toxin framework adjacent to the domain II loops, and (3) a series of overlap extension PCRs to join the recovered CDR-H3 sequences and the toxin framework sequences. Primers, restriction sites, and DNA fragments relevant to the assembly are labeled.