Tandem nanobody: A feasible way to improve the capacity of affinity chromatography

Yunxiang Huang; Jinheng Fu; Yinfeng Zhong; Wenyuan Shuai; Hang Zhang; Yanping Li; Qinghua He; Zhui Tu

doi:10.1016/j.jchromb.2021.122678

Tandem nanobody: A feasible way to improve the capacity of affinity chromatography

J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Mar 29:1173:122678. doi: 10.1016/j.jchromb.2021.122678. Online ahead of print.

Authors

Yunxiang Huang¹, Jinheng Fu², Yinfeng Zhong¹, Wenyuan Shuai³, Hang Zhang³, Yanping Li⁴, Qinghua He⁴, Zhui Tu⁵

Affiliations

¹ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; School of Life Sciences, Nanchang University, Nanchang 330031, China; Jiangxi-OAI Joint Research Institution, Nanchang University, Nanchang 330047, China.
² State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; Jiangxi-OAI Joint Research Institution, Nanchang University, Nanchang 330047, China.
³ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
⁴ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; Jiangxi-OAI Joint Research Institution, Nanchang University, Nanchang 330047, China; Jiangxi Province Key Laboratory of Modern Analytical Sciences, Nanchang University, Nanchang 330031, China.
⁵ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China; Jiangxi Province Key Laboratory of Modern Analytical Sciences, Nanchang University, Nanchang 330031, China. Electronic address: tuzhui@ncu.edu.cn.

PMID: 33839466
DOI: 10.1016/j.jchromb.2021.122678

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. The highest improvement of the static binding capacity was achieved 73.7% by the H-diAFV resin other than the H-triAFV or H-tetAFV, as compared to the H-AFV resin.

Keywords: Affinity chromatography; HaloTag; IgG purification; Tandem Nanobody; anti-Fc.