Peptidisc-assisted hydrophobic clustering towards the production of multimeric and multispecific nanobody proteins

Protein multimerization is a powerful engineering strategy for enhancing structural stability, diversity and functional performance. Typical methods to cluster proteins include tandem linking, fusion to self-assembly domains and cross-linking. We present here an approach that leverages the peptidisc membrane mimetic to stabilize hydrophobic-driven protein associations. We apply the method to nanobodies (Nbs), effective substitutes to antibodies due to their production efficiency, cost effectiveness, and lower immunogenicity, and we demonstrate the formation of multimeric assemblies termed "polybodies" (Pbs). Starting with Nbs directed against the green fluorescent protein (GFP), we produce Pbs that display increased affinity for GFP due to the avidity effect. The benefit of avidity in affinity-based assays is also demonstrated using moderate-affinity Nbs against human serum albumin. With the same auto-assembly principle, we produce bispecific and auto-fluorescent Pbs, validating our method as a versatile and general engineering strategy to generate multispecific and multifunctional protein entities. Peptidisc-assisted hydrophobic clustering thus expands the protein engineering toolbox to broaden the scope of protein multimerization in life sciences. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/630897v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@15b9667org.highwire.dtl.DTLVardef@1ef5132org.highwire.dtl.DTLVardef@bbd090org.highwire.dtl.DTLVardef@79c54b_HPS_FORMAT_FIGEXP M_FIG C_FIG