New Strategy for High-Level Expression and Purification of Biologically Active Monomeric TGF-β1/C77S in Escherichia coli

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• 作者：Yana V. Kim ; Marine E. Gasparian ; Eduard V. Bocharov…
• 关键词：TGF ; β1 ; Monomeric TGF ; β1 ; TGF ; β1/C77S ; Thioredoxin ; tag fusion protein ; Escherichia coli ; Refolding ; CD spectrum
• 刊名：Molecular Biotechnology
• 出版年：2015
• 出版时间：February 2015
• 年：2015
• 卷：57
• 期：2
• 页码：160-171
• 全文大小：1,593 KB
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• 刊物主题：Biotechnology; Biochemistry, general; Cell Biology; Protein Science; Biological Techniques; Human Genetics;
• 出版者：Springer US
• ISSN：1559-0305

文摘

Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5?g?l?) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8?mg from 100?ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.