Tseng Yao-Kuang, Lu Yun-Heng, Liu Yun, Weng Zhi-Wei, Lin Yu-Tzu, Tsai Chih-Hsuan, Wu Yueh-Lung, Huang Rong-Nan
Department of Entomology, National Taiwan University, Taipei, Taiwan.
Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan.
Microb Biotechnol. 2025 May;18(5):e70157. doi: 10.1111/1751-7915.70157.
Efficient and economical purification methods are crucial for the commercial production of recombinant proteins with biomedical applications. In this study, we developed an affinity chromatography system that leverages the polysaccharide-binding properties of galectin-1 (GAL1) as a protein tag. The known GAL1-binding material, chitin, was used as the purification matrix. Melittin (MELT), a bee venom peptide known for its antimicrobial and anti-inflammatory properties with commercial potential, was chosen to validate this system. The GAL1-MELT fusion protein was expressed in Escherichia coli (E. coli) and successfully purified using a chitin-based matrix with sodium dodecyl sulfate (SDS) as a removable eluant. This method demonstrated higher purification efficiency compared to the His-tag/Ni-NTA approach, indicating that the GAL1/chitin system could serve as a superior alternative. The GAL1-MELT fusion protein retained strong antibacterial and anti-inflammatory activities, as well as collagen content modulation effects, confirming that MELT maintained its bioactivity. Apart from that, the GAL1-DsRed fusion protein was used as an additional protein target to evaluate the efficiency of the chitin-based column. Notably, all experiments were conducted without tag cleavage, showing that enzyme treatments for MELT isolation were unnecessary. This study highlights the potential of GAL1-polysaccharide interactions as a cost-effective and highly efficient alternative method for recombinant protein purification.
高效且经济的纯化方法对于具有生物医学应用的重组蛋白的商业化生产至关重要。在本研究中，我们开发了一种亲和色谱系统，该系统利用半乳糖凝集素-1（GAL1）的多糖结合特性作为蛋白质标签。已知的GAL1结合材料几丁质被用作纯化基质。蜂毒肽（MELT）是一种具有抗菌和抗炎特性且具有商业潜力的蜂毒肽，被选来验证该系统。GAL1-MELT融合蛋白在大肠杆菌（E. coli）中表达，并使用基于几丁质的基质以十二烷基硫酸钠（SDS）作为可去除洗脱剂成功纯化。与His标签/Ni-NTA方法相比，该方法显示出更高的纯化效率，表明GAL1/几丁质系统可以作为一种更优的替代方法。GAL1-MELT融合蛋白保留了强大的抗菌和抗炎活性以及胶原蛋白含量调节作用，证实MELT保持了其生物活性。除此之外，GAL1-DsRed融合蛋白被用作另一种蛋白质靶标来评估基于几丁质的柱的效率。值得注意的是，所有实验均在不进行标签切割的情况下进行，表明无需对MELT进行酶处理来分离。本研究突出了GAL1-多糖相互作用作为一种经济高效的重组蛋白纯化替代方法的潜力。
