Mullaney Julienne M, Black Lindsay W
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2014;1108:69-85. doi: 10.1007/978-1-62703-751-8_5.
Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.
由于其物理弹性和基因组灵活性，噬菌体T4已证明自身易于用于基于噬菌体的DNA和蛋白质包装、表达及展示系统。作为一种具有可去除内部蛋白和可去除外衣壳蛋白的大型双链DNA噬菌体，它可被改造用于在衣壳内包装感兴趣的DNA和蛋白质，并在衣壳外部展示肽和蛋白质。任何序列的单个170 kb线性DNA，或较短线性DNA的单拷贝或多拷贝，均可在体外由大型末端酶亚基包装到含蛋白质的原头部中，并形成完整或部分完整的衣壳。用于DNA包装的原头部容器还可展示来自衣壳展示蛋白HOC和SOC的肽或全长蛋白。我们实验室还开发了一种蛋白质表达、包装和加工（PEPP）系统，我们发现该系统相对于哺乳动物和细菌细胞系统具有优势，包括高产率、更高的稳定性以及简化的下游加工。我们通过噬菌体PEPP平台生产的蛋白质包括人类HIV-1蛋白酶、来自金黄色葡萄球菌的微球菌内切酶、限制性内切酶EcoRI、荧光素酶、人类粒细胞集落刺激因子（GCSF）、绿色荧光蛋白（GFP）以及淀粉样前体蛋白（APP）的99个氨基酸的C末端。在哺乳动物蛋白质表达系统中难以生产的有毒蛋白质，利用PEPP平台可轻松生产、包装和加工。APP就是这样一种已成功生产的极难处理的蛋白质的一个例子。以下方法描述了用DNA体外包装原头部以及生产携带噬菌体基因组中感兴趣基因并产生和在内部包装相应感兴趣蛋白质的重组T4噬菌体的步骤。
