Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme.
We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting nonoverlapping epitopes; individual binders of KD∼ 1.3 μM and 250 nM produced a bivalent binder with apparent KD∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (TM= 84.5 °C) and high recombinant expression yields in E. coli. Finally, when binders comprising the bivalent protein are fused to two of the three fragments of tripartite split-green fluorescent protein (GFP), target-dependent reconstitution of fluorescence occurs, thereby enabling a "mix-and-read" assay for target quantification.
Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
Kevin B
KB
Carlin
Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
Carlos A
CA
Cruz-Teran
Institute of Stem Cell Biology and Regenerative Medicine, NCBS-TIFR , Bangalore 560065, Karnataka, India.|School of Chemical & Biotechnology, SASTRA University , Thanjavur 613401, Tamil Nadu, India.
Jay Prakash
JP
Kumar
Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
Catherina
C
Gomes
Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
Balaji M
BM
Rao
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; C093228:Sso7d protein, Sulfolobus; D049452:Green Fluorescent Proteins
Sso7d; binding affinity; flexible linker; split GFP; yeast surface display