Du Jinping, Rehm Bernd H A
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, Brisbane, Australia.
Biotechnol Lett. 2018 Feb;40(2):369-373. doi: 10.1007/s10529-017-2473-4. Epub 2017 Nov 9.
To overcome laborious and costly procedures often associated with therapeutic protein production and purification, in vivo polyester immobilized sortase is explored for the production of human tumor necrosis factor alpha (TNFα) and human interferon alpha 2b (IFNα2b) by Escherichia coli.
Hybrid genes encoding PhaC-Sortase-TNFα or PhaC-Sortase-IFNα2b fusions (with a LPETG recognition signal immediately before TNFα or IFNα2b), mediated intracellular production of polyester (polyhydroxyalkanoate, PHA) beads in Escherichia coli. Upon isolation of respective PHA beads, pure soluble TNFα or IFNα2b was released by activating sortase via addition of CaCl and triglycine. TNFα and IFNα2b each were recognized by corresponding conformational antibodies in an ELISA assay.
In vivo polyester immobilized sortase could be exploited for production and purification of high-value therapeutic proteins without laborious and costly downstream processing.
为克服与治疗性蛋白质生产和纯化相关的繁琐且成本高昂的程序，探索利用体内聚酯固定化分选酶通过大肠杆菌生产人肿瘤坏死因子α（TNFα）和人干扰素α2b（IFNα2b）。
编码PhaC-分选酶-TNFα或PhaC-分选酶-IFNα2b融合体（在TNFα或IFNα2b之前紧接LPETG识别信号）的杂交基因介导了大肠杆菌中聚酯（聚羟基脂肪酸酯，PHA）珠粒的细胞内生产。分离出各自的PHA珠粒后，通过添加氯化钙和三甘氨酸激活分选酶，释放出纯的可溶性TNFα或IFNα2b。在酶联免疫吸附测定中，TNFα和IFNα2b各自被相应的构象抗体识别。
体内聚酯固定化分选酶可用于生产和纯化高价值治疗性蛋白质，而无需繁琐且成本高昂的下游加工。
