Related Experiment Video
Updated: Jan 28, 2026

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
Two-in-one protein tag enables the soluble expression and calcium-dependent purification of single-chain antibody
Jonghwan Lee1,2, Heesun Park1, Suhyeon Jeong1
1Center for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering and Technology, Cheongju, Republic of Korea.
Abstract:
Single-chain variable fragments (scFvs) antibodies are used in diagnostic and therapeutic biopharmaceuticals. However, its production is limited due to insoluble expression and lack of a universal purification ligand. Here, we report a novel two-in-one protein tag, termed CSQ-tag, utilizing a calsequestrin (CSQ) protein to enable the soluble expression and calcium-dependent purification for scFvs. The CSQ-tag enhanced the solubility of four different therapeutic scFvs with an average solubility of 83.8 ± 4.6% in the BL21(DE3) strain, despite its reducing cytoplasmic environment. Compared to other tags, CSQ-tag showed superior solubility, with 1.8-fold higher solubility than MBP-tag across four scFvs. The solubility enhancement of the CSQ-tag is attributed to two main characteristics: highly acidic sequences with a low isoelectric point of 3.95, and intrinsically disordered protein properties. Furthermore, CSQ-tag can cost-effectively purify therapeutic scFvs with high purity over 95% using the calcium-dependent phase transition properties. Importantly, the anti-VEGF-scFv produced with the CSQ-tag showed a binding affinity of 25.8 pM, similar to that of the existing therapeutic brolucizumab. Therefore, CSQ-tag can significantly improve productivity and simultaneously maintain scFvs' functional integrity, thereby serving as a promising tool for universal expression and purification of therapeutic scFvs.
Related Concept Videos
Factors Affecting Solubility
Solubility Equilibria
The...
Mass Spectrometry: Long-Chain Alkane Fragmentation
Solubility of Ionic Compounds
Tagging and Fusion Proteins
Antibody Structure
Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
The Y-Shaped Structure of Antibodies Consists of Four Polypeptide Chains
Antibodies consist of four polypeptide chains: two identical heavy...

