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.