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.
Microb Cell Fact. 2017 Nov 2;16(1):184. doi: 10.1186/s12934-017-0799-1.
Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction.
By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).
Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.
从大肠杆菌中重组蛋白的生产和纯化通常伴随着昂贵和复杂的程序，特别是对于治疗性蛋白。在这里，通过使用可切割内含肽的聚羟基烷酸合酶融合物，首先可以生产并隔离重组蛋白，将其隔离在天然树脂聚羟基烷酸（PHA）包涵体中，然后通过简单的 PHA 珠分离步骤从污染的宿主蛋白中分离出来，最后通过特定的 pH 值降低诱导可溶性部分的释放进行纯化。
通过使用自我切割内含肽将目标蛋白翻译融合到 PHA 合酶上，实现了 PHA 珠的细胞内生产。通过分离相应的 PHA 珠，可以通过简单的 pH 值降至 6 来释放可溶性的纯目标蛋白。通过生产六种目标蛋白，包括维多利亚水母绿色荧光蛋白（GFP）、结核分枝杆菌候选疫苗 Rv1626、金黄色葡萄球菌蛋白 A 的 IgG 结合 ZZ 结构域、人肿瘤坏死因子-α（TNFα）、人粒细胞集落刺激因子（G-CSF）和人干扰素α 2b（IFNα2b），证明了这种方法的实用性。
这里开发了一种通过可切割内含肽的聚羟基烷酸合酶融合物生产和纯化无标签蛋白的新方法。无需繁琐的下游处理即可轻松获得纯目标蛋白。
