Tandem Nanobody: a feasible way to improve the capacity of affinity
chromatography
Abstract
Nanobodies, referred to the binding domain of the heavy-chain-only
antibodies, are the smallest antigen recognition unit. The molecular
weight of monomeric nanobodies is about one-tenth of the conventional
antibodies. The small size of nanobodies facilitates genetic
manipulation and recombinant expression. This study aimed to investigate
the effects of nanobody multivalency on the binding capacity of affinity
resin. The nanobody (namely AFV), which binds to the fragment
crystallizable (Fc) region of immunoglobulin G (IgG), was fused to the
N-terminal of HaloTag in the form of monomeric (H-AFV), dimer (H-diAFV),
trimer (H-triAFV), and tetramer (H-tetAFV). The fusion proteins were
solubly expressed in Escherichia coli yielding at least 9.9 mg L-1. The
biolayer interferometry confirmed an increment of avidity as the
increase of AFV valences. The four recombinant proteins in crude cell
lysate were site-specifically immobilized onto the Halo ligand resin via
the self-labeling HaloTag, respectively. The generated affinity resins
were able to isolate high purity IgG from mouse plasma. An improvement
of 73.7% of the static binding capacity was achieved by the H-diAFV
resin as compared to the H-AFV affinity resin.