The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, School of Life Science, Fujian Agriculture and Forestry University, Fuzhou, China.
Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/College of Crop Science, Fujian Agriculture and Forestry University, Jinshan, Fuzhou, 350002, China.
Protein J. 2019 Dec;38(6):675-682. doi: 10.1007/s10930-019-09855-2.
Thymosin beta 10 (TB10) is one of the common members among beta-thymosins. Human TB10 is reported to play a role in anti-angiogenesis and inhibition of cell migration during the tumorigenesis or metastasis of some certain cancers. Thus, it would be a potent clinical agent. In the present study, the coding sequence of TB10 was optimized based on the codon preference of Escherichia coli and cloned to pET28a (+) by chemical synthesis and molecular cloning methods. The recombinant protein was highly expressed employing E. coli expressing system and purified by a simple step of Ni affinity chromatography. The TEV proteinase recognition site was inserted in the His-tag and the target protein for easy removal of the His-tag. To improve the biological activity of TB10, the transactivator of transcription (TAT) short peptide, a transduction domain, was added to the N-terminus of TB10. About 14.3 mg of the recombinant TB10 proteins was obtained from 1 L bacterial culture. The functional analyses demonstrated that the recombinant TB10 proteins displayed the distinct inhibition on angiogenesis by chick embryo chorioallantoic membrane assay and endothelial cell migration by wound healing assay. The TAT-fused TB10 even had stronger effects, probably due to the better transduction into the cells.
胸腺素β 10（TB10）是β-胸腺素家族中的常见成员之一。据报道，人 TB10 在某些癌症的发生或转移过程中发挥着抗血管生成和抑制细胞迁移的作用。因此，它将成为一种有效的临床药物。在本研究中，根据大肠杆菌的密码子偏好性，对 TB10 的编码序列进行了优化，并通过化学合成和分子克隆方法将其克隆到 pET28a（+）上。采用大肠杆菌表达系统进行重组蛋白的高效表达，并通过简单的 Ni 亲和层析进行纯化。在 His 标签和目的蛋白中插入了 TEV 蛋白酶识别位点，便于去除 His 标签。为了提高 TB10 的生物学活性，在 TB10 的 N 端添加了转录激活因子（TAT）短肽，即转导结构域。从 1L 细菌培养物中获得了约 14.3mg 的重组 TB10 蛋白。功能分析表明，重组 TB10 蛋白在鸡胚绒毛尿囊膜试验中表现出明显的抑制血管生成作用，在伤口愈合试验中表现出抑制内皮细胞迁移的作用。融合了 TAT 的 TB10 甚至具有更强的作用，这可能是由于更好地转导进入细胞。
