ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces
| Autor: | Víctor de Lorenzo, Yamal Al-Ramahi, Ákos Nyerges, Györgyi Ferenc, Lidia Cerdán, Csaba Pál, Yago Margolles, Luis Ángel Fernández |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
DNA
Bacterial QH301-705.5 Medicine (miscellaneous) Mutagenesis (molecular biology technique) DNA Single-Stranded Complementarity determining region Bacterial genome size medicine.disease_cause General Biochemistry Genetics and Molecular Biology Epitope Article Recombineering Applied microbiology 03 medical and health sciences 0302 clinical medicine Antigen In vivo medicine Escherichia coli Biology (General) Gene 030304 developmental biology Intimin 0303 health sciences biology Oligonucleotide Chemistry Nanobody diversification recombineering DIvERGE translocated intimin receptor Single-Domain Antibodies Antibodies Bacterial Cell biology Genetic engineering biology.protein Antibody General Agricultural and Biological Sciences Bacterial outer membrane 030217 neurology & neurosurgery |
| Zdroj: | Communications Biology Communications Biology, Vol 4, Iss 1, Pp 1-11 (2021) |
| DOI: | 10.1101/2021.01.28.428624 |
| Popis: | ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. 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. 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. 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. Yamal Al-ramahi et al. describe an accelerated laboratory evolution method that applies ssDNA recombineering and bacterial surface display to engineer nanobody epitope recognition. As a proof-of-concept, they evolved TD4 recognition to bind the translocated intimin receptor (TirM) of EPEC pathogens, with this method bypassing the need for laborious cloning and mutagenesis procedures. |
| Databáze: | OpenAIRE |
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