Fibronectin type III domain based monobody with high avidity.
Significant efforts have been made to improve the target-binding strength of numerous affinity molecules that are widely used in biomedical research and clinical applications. While many antibody-like fragments have been successfully optimized through affinity maturation, the process is time-consuming and restricted by the stability, solubility, and expression level of the protein sequences. To generate a fibronectin type III domain (FN3) based monobody that binds to the tumor-related biomarkers with exceptional high strength and stability, we developed a multivalent strategy by fusing an alphavbeta3-binding FN3 monobody with a short COMP pentamerization domain through a linker that facilitates appropriate display of multivalent FN3 domains. The fusion protein was highly expressed in the soluble fraction of Escherichia coli and efficiently self-assembled into a pentameric molecule that could be readily purified. Compared to the monomeric form, the pentameric monobody bound to alphavbeta3 integrin much more tightly with significantly slower off-rate, while still maintaining its excellent specificity toward alphavbeta3 when tested by using purified integrins and integrin-expressing cell lines. The multivalent strategy we describe here could be applied to engineer other FN3 monobodies to acquire significantly improved targeting-binding strengths.
Duan
Jinzhu
J
School of Pharmacy and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Wu
Jinsong
J
Valencia
C Alexander
CA
Liu
Rihe
R
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000915:Antibody Affinity; D019281:Dimerization; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000276:immunology*; D020014:K562 Cells; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D039302:Integrin alphaVbeta3; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases