Intein-mediated expression, purification, and characterization of thymosin 伪1-thymopentin fusion peptide in Escherichia coli

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• 作者：Juan Li^a ; Lei Zheng^b ; Pingli Li^a ; Fengshan Wang^a ; ^c ; ^{fswang@sdu.edu.cn} ; ^{wangfengshansdu@hotmail.com}
• 关键词：Thymosin 伪1&ndash ; thymopentin ; Intein ; Self-cleavage ; Protein refolding ; ESI-MS ; Immunoregulatory activity
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：July, 2012
• 卷：84
• 期：1
• 页码：1-8
• 文件大小：775 K

摘要

Thymosin 伪1-thymopentin (T伪1-TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than T伪1 and TP5. To obtain T伪1-TP5 more effectively and economically, T伪1-TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin beads in Escherichia coli. After affinity purification, the target peptide was released from the chitin beads via self-cleaving intein ((INTervening protEIN) induced by dithiothreitol. Further, T伪1-TP5 was purified by Superdex 30 and identified by Tricine-SDS-PAGE and electrospray ionization-mass spectrometry. Finally, about 7.6 mg T伪1-TP5 purified from the soluble fraction and inclusion bodies was obtained from 1 L culture media. The purity was 95%after a series of chromatographic purification steps. In vitro, the purified T伪1-TP5 could stimulate the proliferation of mouse splenic lymphocytes. Overall, this work demonstrated that T伪1-TP5 was purified with low cost and high efficiency, greatly expanding its potential use as an immune regulator.