Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham, Plant Biotechnology R and D Center, Shanghai Jiao Tong University, Shanghai 200240, China.
Biomed Res Int. 2013;2013:241721. doi: 10.1155/2013/241721. Epub 2013 May 19.
Thymosin β4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termed Tβ4) was designed based on the Tβ4 protein sequence and used to express a 4 × Tβ 4 concatemer (four tandem copies of Tβ4, termed 4 × Tβ4) together with a histidine tag (6 × His) in E. coli (strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl β-D-thiogalactoside (IPTG). The E. coli-derived 4 × Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cells in vitro and in vivo wound healing. The results demonstrate that these activities of the E. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.
胸腺肽 β4（Tβ4）是最有前途的胸腺肽之一，预计未来对 Tβ4 的商业需求将会增加。为了开发生产重组 Tβ4 的新方法，根据 Tβ4 蛋白序列设计了一段 168bp 的 DNA（称为 Tβ4），用于在大肠杆菌（BL21 菌株）中表达 4×Tβ4 串联体（4 个 Tβ4 串联拷贝，称为 4×Tβ4）和组氨酸标签（6×His）。SDS-PAGE 和 Western blot 分析证实，在诱导细菌培养物中使用异丙基 β-D-硫代半乳糖苷（IPTG）后，可产生预期大小（30.87kDa）的重组 4×Tβ4 蛋白。通过 Ni-NTA 树脂纯化大肠杆菌来源的 4×Tβ4，并检查其体外刺激小鼠脾细胞增殖和体内伤口愈合的活性。结果表明，该大肠杆菌来源的重组 4×Tβ4 的这些活性与现有商业获得的 Tβ4 相似甚至更好。因此，这种生产策略代表了未来重组 Tβ4 商业生产的一种有价值的方法。
