2. Academic Validation
  3. Structure-guided design of a minimal epitope tag and nanobody affinity system

Structure-guided design of a minimal epitope tag and nanobody affinity system

  • J Chromatogr A. 2026 Feb 8:1768:466675. doi: 10.1016/j.chroma.2026.466675.
Jiayi Tan 1,  Limin Zhao 2,  Mu Li 3,  Kanglong Yang 4,  Yue Li 5,  Yuqing Ding 6,  Heng Liu 3,  Houjun Xia 7,  Chongyuan Wang 8,  Jiawei Li 9
Affiliations

Affiliations

  • 1 Department of Geriatric Medicine, Shenzhen Longhua District Central Hospital, Shenzhen 518110, China; Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 581055, China; College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China; Faculty of Pharmaceutical Sciences, Shenzhen University of Advanced Technology, Shenzhen 518055, China.
  • 2 Department of Stomatology, Shenzhen Guangming District People's Hospital, Shenzhen, 518106, China.
  • 3 The Affiliated Traditional Chinese Medicine Hospital, GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, State Key Laboratory of Respiratory Disease, Guangzhou 511436, China.
  • 4 Department of Cardiovascular Medicine, Shenzhen Longhua District Central Hospital, Shenzhen 518110, China.
  • 5 Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
  • 6 Faculty of Pharmaceutical Sciences, Shenzhen University of Advanced Technology, Shenzhen 518055, China.
  • 7 Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 581055, China; Faculty of Pharmaceutical Sciences, Shenzhen University of Advanced Technology, Shenzhen 518055, China. Electronic address: [email protected].
  • 8 Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 581055, China; Faculty of Pharmaceutical Sciences, Shenzhen University of Advanced Technology, Shenzhen 518055, China. Electronic address: [email protected].
  • 9 Department of Geriatric Medicine, Shenzhen Longhua District Central Hospital, Shenzhen 518110, China; Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 581055, China. Electronic address: [email protected].
PMID: 41512648 DOI: 10.1016/j.chroma.2026.466675
Abstract

The isolation of recombinant proteins commonly relies on affinity-based methods. Nevertheless, widely used tags and solid supports are frequently constrained by insufficient specificity, harsh elution requirements, and cost inefficiency. Here, we developed a robust purification system based on a nanobody that specifically recognizes a short peptide epitope. The nanobody is produced at high yield in a prokaryotic expression system, and the corresponding peptide tag, comprising only 6-7 Amino acids, can be fused to either the N- or C-terminus of target proteins. We demonstrate that this system enables one-step purification of diverse soluble and membrane proteins from cell lysates, with high purity and yield. Bound proteins are gently eluted under non-denaturing conditions using a competitive synthetic peptide, achieving a recovery yield of over 90%. Furthermore, the nanobody-conjugated resin maintains consistent binding capacity over at least 10 reuse cycles. Given its high specificity, mild elution, reusability, and simplicity, we propose this system as a versatile and efficient platform for high-quality Protein Purification, with broad applicability in proteomics, structural biology, and biotherapeutic development.

Keywords

Affinity tag; Fe-tag; Nanobody; Protein purification; Structural biology.

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