neb 10 beta electrocompetent cells  (New England Biolabs)

Journal: Bio-protocol

Article Title: Isolation of Antigen-Specific Nanobodies From Synthetic Libraries Using a Protein Selection Strategy That Combines MACS-Based Screening of YSD and FLI-TRAP

doi: 10.21769/BioProtoc.5570

Figure Lengend Snippet: A synthetic nanobody library is displayed on the surface of Saccharomyces cerevisiae BJ5465 and subjected to antigen-specific enrichment using MACS with recombinant PCSK9. This step selectively enriches yeast clones expressing Nbs with binding activity toward PCSK9. The enriched Nb population is subsequently transferred to a bacterial functional screening system based on functional ligand-binding identification by twin-arginine translocation (Tat)-based recognition of associating proteins (FLI-TRAP). In the FLI-TRAP system, Nb candidates are expressed in the cytoplasm of Escherichia coli as N-terminal fusions to the Tat signal peptide derived from trimethylamine N-oxide reductase (ssTorA), whereas PCSK9 is expressed as a fusion with the reporter enzyme β-lactamase (Bla). Productive Nb–PCSK9 interactions in the cytoplasm result in the formation of a stable protein complex that is specifically recognized by the Tat translocase. The Tat pathway mediates the translocation of fully folded proteins and protein complexes across the inner membrane into the periplasm; consequently, only correctly folded and sufficiently stable Nb–PCSK9–Bla complexes are exported. Periplasmic localization of Bla confers resistance to β-lactam antibiotics, thereby enabling direct in vivo selection of PCSK9-specific Nbs with favorable binding affinity and solubility based on bacterial survival under antibiotic selection pressure.

Article Snippet: Competent NEB10β E. coli cells (New England Biolabs, catalog number: C3019H) 3.

Techniques: Recombinant, Clone Assay, Expressing, Binding Assay, Activity Assay, Functional Assay, Ligand Binding Assay, Translocation Assay, Derivative Assay, Membrane, In Vivo, Selection, Solubility