Zhang Jie, Ma Lei, Zhang Shuang Quan
Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou, Jiangsu Province, PR China; Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Science College, Nanjing Normal University, Nanjing 210046, Jiangsu, PR China.
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Science College, Nanjing Normal University, Nanjing 210046, Jiangsu, PR China.
Protein Expr Purif. 2014 Mar;95:177-81. doi: 10.1016/j.pep.2013.12.013. Epub 2014 Jan 10.
APRIL is a member of the tumor necrosis factor (TNF) family of ligands that mediate tumor cells proliferation as well as survival, depending on the cellular context. In this report, we present a novel method to obtain soluble human APRIL in Escherichia coli using the elastin-like polypeptide and SUMO (ELP-SUMO) tags. The fusion protein with ELP-SUMO tag was expressed in a soluble form at 15°C. After purification based on inverse transition cycling (ITC) method, the purified ELP-SUMO-hAPRIL fusion protein was subsequently cleaved by SUMO protease to release mature hAPRIL. Following affinity chromatography, the target protein was re-purified with high purity. Finally, about 4.8mg recombinant hAPRIL was obtained from 1l bacterial culture with no less than 85% purity. The molecular mass (Mr) of the recombinant hAPRIL was confirmed by MALDI-TOF MS as Mr 16,314. The purified hAPRIL exhibits biological activity on Jurkat cells. It is the first report on soluble production of hAPRIL in E. coli using ELP-SUMO tag.
APRIL是肿瘤坏死因子（TNF）配体家族的成员，根据细胞环境介导肿瘤细胞的增殖和存活。在本报告中，我们提出了一种利用弹性蛋白样多肽和SUMO（ELP-SUMO）标签在大肠杆菌中获得可溶性人APRIL的新方法。带有ELP-SUMO标签的融合蛋白在15°C时以可溶性形式表达。基于逆转变循环（ITC）方法纯化后，纯化的ELP-SUMO-hAPRIL融合蛋白随后被SUMO蛋白酶切割以释放成熟的hAPRIL。经过亲和层析后，目标蛋白以高纯度重新纯化。最后，从1升细菌培养物中获得了约4.8毫克重组hAPRIL，纯度不低于85%。重组hAPRIL的分子量（Mr）通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）确认为Mr 16,314。纯化的hAPRIL对Jurkat细胞具有生物学活性。这是首次关于使用ELP-SUMO标签在大肠杆菌中可溶性生产hAPRIL的报道。
