Yin Shuang, Zhang Bingyang, Lin Jianying, Liu Yongdong, Su Zhiguo, Bi Jingxiu
School of Chemical Engineering & Advanced Materials Faculty of Engineering, Computer and Mathematical Sciences University of Adelaide Adelaide Australia.
College of Biomedical Engineering Taiyuan University of Technology Taiyuan P. R. China.
Eng Life Sci. 2021 Jun 19;21(10):630-642. doi: 10.1002/elsc.202000105. eCollection 2021 Oct.
Ferritin is a promising drug delivery platform and has been functionalized through genetic modifications. This work has designed and expressed a dual-functional engineered human heavy-chain ferritin (HFn) with the inserted functional peptide PAS and RGDK to extend half-life and improve tumor targeted drug delivery. A facile and cost-effective two-step purification pathway for recombinant HFn was developed. The genetic modification was found to affect HFn conformation, and therefore varied the purification performance. Heat-acid precipitation followed by butyl fast flow hydrophobic interaction chromatography (HIC) has been developed to purify HFn and modified HFns. Nucleic acid removal reached above 99.8% for HFn and modified HFns. However, HFn purity reached above 95% and recovery yield (overall) above 90%, compared with modified HFns purity above 82% and recovery yield (overall) above 58%. It is interesting to find that the inserted functional peptides significantly changed the molecule conformation, where a putative turnover of the E-helix with the inserted functional peptides formed a "flop" conformation, in contrast with the "flip" conformation of HFn. It could be the cause of fragile stability of modified HFns, and therefore less tolerant to heat and acid condition, observed by the lower recovery yield in heat-acid precipitation.
铁蛋白是一种很有前景的药物递送平台，并且已通过基因改造实现了功能化。这项工作设计并表达了一种具有插入功能肽PAS和RGDK的双功能工程化人重链铁蛋白（HFn），以延长半衰期并改善肿瘤靶向药物递送。开发了一种简便且经济高效的重组HFn两步纯化途径。发现基因改造会影响HFn的构象，因此改变了纯化性能。已开发出先进行热酸沉淀，然后进行丁基快速流动疏水相互作用色谱（HIC）的方法来纯化HFn和修饰后的HFn。HFn和修饰后的HFn的核酸去除率达到99.8%以上。然而，HFn的纯度达到95%以上，总回收率达到90%以上，相比之下，修饰后的HFn纯度在82%以上，总回收率在58%以上。有趣的是，发现插入的功能肽显著改变了分子构象，其中插入功能肽后E螺旋的推定翻转形成了“耷拉”构象，这与HFn的“翻转”构象形成对比。这可能是修饰后的HFn稳定性脆弱的原因，因此在热酸沉淀中观察到其对热和酸条件的耐受性较低，回收率也较低。
