Enhance the affinity of nanobodies to CD47 antigen by fusion expression of COMP48 self-folding peptide

Abstract

Objective To modify CD47 nanobody with the self-folding peptide human cartilage oligomeric matrix protein (COMP48) so as to enhance its affinity to CD47 antigen. Methods The fusion sequences of COMP48 and CD47 nanobody (VHHB1) were designed and synthesized, and the recombinant plasmid pET22b-VHHB1-COMP48 was constructed and transformed into E. coli BL21 (DE3) to induce expression of the fusion protein. The binding specificity and affinity of the fusion protein and the antigen CD47 were detected by Western Blot, indirect enzyme-linked immunosorbent assay (ELISA) and non-competitive ELISA. Results The recombinant VHHB1-COMP48 was expressed in BL21(DE3) by inducing with 1 mmol/L IPTG and purified at 90% homogenous in IMAC. Western Blot results showed that the recombinant protein VHHB1-COMP48 specifically binds to antigen CD47 but not to unrelated protein. The indirect ELISA and non-competitive ELISA results showed that the affinity of the conjugated recombinant protein VHHB1-COMP48 was enhanced compared to that of the non-conjugated nanobody, and the difference was statistically significant (P<0.01). Through non-competitive ELISA, the constants of affinity and dissociation constants were 6.97×107 L/mol and 1.434×10-8 mol/L, respectively. Conclusions The affinity of the nanobody for the antigen can be improved by conjugating a human cartilage matrix protein (COMP48) after the nanobody. Key words: Nanobody; Human cartilage oligomeric matrix protein; Affinity maturation; Self-folding peptide; CD47