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 Ni²⁺ affinity chromatography. The TEV proteinase recognition site was inserted in the His₆-tag and the target protein for easy removal of the His₆-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.

中文翻译：

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TAT融合胸腺素β10的重组表达和生物活性鉴定。

胸腺素β10（TB10）是β-胸腺素中的常见成员之一。据报道，人TB10在某些某些癌症的发生或转移过程中在抗血管生成和抑制细胞迁移中起作用。因此，它将是有效的临床药物。在本研究中，基于大肠杆菌的密码子偏好性优化了TB10的编码序列，并通过化学合成和分子克隆方法将其克隆到pET28a（+）。利用大肠杆菌表达系统高表达重组蛋白，并通过简单的Ni ²⁺亲和层析步骤纯化。将TEV蛋白酶识别位点插入His ₆标签中，并插入目标蛋白以轻松去除His ₆-标签。为了提高TB10的生物活性，将转录反式激活子（TAT）短肽（一种转导域）添加到TB10的N端。从1L细菌培养物中获得约14.3mg的重组TB10蛋白。功能分析表明，重组TB10蛋白通过鸡胚绒膜尿囊膜测定和伤口愈合测定显示出对血管生成的明显抑制作用。TAT融合的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所致。