On-Column Dual-Gradient Refolding for Efficient Recovery of Insoluble Affinity-Tagged Recombinant Proteins

BIO-PROTOCOL 2026 Open access

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Authors: Anna Vlaskina, Dmitry E. Petrenko, Yulia К. Agapova, A.A. Kuzminkova, Martha A. Evteeva, Maxim V. Patrushev
DOI: 10.21769/bioprotoc.5598
Cited by: 0

Abstract

protein expression and an FPLC system, being particularly suitable for insoluble proteins that form inclusion bodies. • The method utilizes a dual-gradient refolding strategy on a Ni-TED column, integrating solubilization, refolding, and initial purification into a single workflow. • Effectively rescues challenging proteins from inclusion bodies, demonstrated with aggregation-prone CRISPR-associated effector homologs (Cas12m) refractory to traditional refolding methods. • Utilizes Ni-TED resin for its compatibility with high β-mercaptoethanol concentrations and moderate binding affinity, reducing nonspecific binding of host cell proteins.

Fields of Study & Subjects

Protein purification and stability Enzyme Production and Characterization Protein Structure and Dynamics

Publication details

Journal / source
BIO-PROTOCOL
Volume
16
Issue
1390
Pages
e5598–e5598
Publication date
1 January 2026
Language
EN