A new metal affinity NCTR25 tag as a better alternative to the His-tag for the expression of recombinant fused proteins

Abstract

His-tagging is commonly used in fusion protein production, but the His-tag is usually prohibited in medicinal proteins and must be removed. A fragment (NCTR25-tag) truncated from the N-terminus of human copper transporter 1 was tested for feasibility as a replacement for the His-tag in fusion proteins. The NCTR25-tag and His-tag were separately fused to the transthyretin (TTR) protein, and the expression, affinity purification, refolding and stability of the two kinds of fusions were compared. NCTR25 fusion produced a 63% higher yield of the recombinant protein, which was purified by metal affinity chromatography with an efficiency similar to that of His-tagged protein. NCTR25-tag fusion had much less impact on the foldability, kinetic and thermodynamic stability of tetrameric TTR than His-tag fusion. When the tags were individually fused to enhanced green fluorescent protein (EGFP), NCTR25 fusion yielded 29–128% more product than His-EGFP. NCTR25-EGFP could be purified by metal affinity chromatography and showed better foldability than His-EGFP. Furthermore, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) fusion with the third disulfide loop of TGF-α (TGF3L-TRAIL) fused with the NCTR25-tag retained the stability and superactivity of His-TGF3L-TRAIL. Therefore, the native tag NCTR25-tag is a feasible alternative to the His-tag in medicinal recombinant proteins.