One remarkable feature of this course is that in vivo diversification of the CDR sequences for optimally binding a molecular target is virtually limited to 2–4 residues of the primary protein sequence 5. Such a process occurs upon exposure of the immune system to given antigens through an intricate molecular mechanism of clonal selection, amplification and affinity/specificity maturation 4, 5. While the variable (V) domain of different antibodies adopt a very similar immunoglobulin (Ig) fold structure, their antigen-binding loops (the so-called complementarity determinant regions or CDRs) are selected out of a very large diversity pool 3. The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest.Īntibodies are the best instance of functional biomolecules which owe their performance to the ability of their encoding DNA of combining in vivo a limited number of hypervariable sequences with a virtually fixed protein scaffold 1, 2. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. SsDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties.

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