Method for Preparing Recombinant Galectin-2 Protein without Escherichia coli-Specific Post-translational Modifications

• Published in: Biological & pharmaceutical bulletin 2023/12/01, Vol.46(12), pp.1676-1682
• Main Authors: Tamura, Mayumi, Fujii, Norihiko, Takeuchi, Tomoharu, Tsuyuguchi, Masato, Tanikawa, Takashi, Oka, Saori, Hatanaka, Tomomi, Kishimoto, Seishi, Kato, Ryuichi, Arata, Yoichiro
• Format: Article
• Language: English
• Subjects: Affinity; Affinity chromatography; Chromatography; E coli; Escherichia coli; galectin-2; Gastrointestinal tract; Histidine; Mass spectroscopy; mistranslation; Molecular weight; N-Terminus; phosphogluconoylation; Post-translation; Proteins; recombinant protein; Stop codon; Tryptophan
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b23-00344

Galectin-2 (Gal-2) is an animal lectin with specificity for β-galactosides. It is predominantly expressed and suggested to play a protective function in the gastrointestinal tract; therefore, it can be used as a protein drug. Recombinant proteins have been expressed using Escherichia coli and used to study the function of Gal-2. The recombinant human Gal-2 (hGal-2) protein purified via affinity chromatography after being expressed in E. coli was not completely homogeneous. Mass spectrometry confirmed that some recombinant Gal-2 were phosphogluconoylated. In contrast, the recombinant mouse Gal-2 (mGal-2) protein purified using affinity chromatography after being expressed in E. coli contained a different form of Gal-2 with a larger molecular weight. This was due to mistranslating the original mGal-2 stop codon TGA to tryptophan (TGG). In this report, to obtain a homogeneous Gal-2 protein for further studies, we attempted the following methods: for hGal-2, 1) replacement of the lysine (Lys) residues, which was easily phosphogluconoylated with arginine (Arg) residues, and 2) addition of histidine (His)-tag on the N-terminus of the recombinant protein and cleavage with protease after expression; for mGal-2, 3) changing the stop codon from TGA to TAA, which is commonly used in E. coli. We obtained an almost homogeneous recombinant Gal-2 protein (human and mouse). These results have important implications for using Gal-2 as a protein drug.