通过ubiquitin表达系统单步净化功能性Cas9蛋白
Xinzhi Duan1, Zhengrong Zhou2, Xiaotong Zeng3
1Department of Biology, College of Sciences, Shantou University, Shantou, 515063, China; Neuroscience center, Department of Basic Medical Sciences, Shantou University Medical College, Shantou, Guangdong, 515041, China.
International journal of biological macromolecules
|September 13, 2025
概括
本研究提出了一种简化,单步净化方法,用于生产高度纯净和功能性的Streptococcus pyogenes Cas9 (SpCas9) 蛋白质. 这种高效的Ubiquitin-Cas9 (Ub-Cas9) 生产有助于广泛的基因编辑应用.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 基因组学就是基因组学.
背景情况:
- 克里斯普尔/卡斯9是精确基因组编辑的关键技术.
- 克里斯普尔/卡斯9核糖蛋白 (RNP) 复合体提供高编辑效率.
- 现有的Cas9蛋白质生产涉及复杂的,多步骤的净化.
研究的目的:
- 为Streptococcus pyogenes Cas9 (SpCas9) 开发一个简化的一步净化策略.
- 为了高效地生产高度纯净和功能性的SpCas9蛋白质.
- 为了使SpCas9在基因编辑研究中得到更广泛的应用.
主要方法:
- 在大肠杆菌中化SpCas9的N终端Ubiquitin (Ub) 的诱导表达.
- 单阶段金属亲和色谱用于净化.
- 用于体外和体内验证的内核酶活性测定.
主要成果:
- 实现了超过8mg/L的95%纯的Ub-Cas9.9的生产.
- 乌比奎丁融合维持了SpCas9的核定位和蛋白质稳定性.
- 纯化Ub-Cas9显示出强大的DNA裂变能力.
结论:
- 建立了一种简化和高效的方法来生产高纯度的Ub-Cas9蛋白质.
- 这种方法提高了SpCas9在研究中的可访问性和实用性.
- 精简的生产方便在基因功能研究中广泛使用.
相关概念视频
Factors Affecting Solubility
39.4K
Compared with pure water, the solubility of an ionic compound is less in aqueous solutions containing a common ion (one also produced by dissolution of the ionic compound). This is an example of a phenomenon known as the common ion effect, which is a consequence of the law of mass action that may be explained using Le Chȃtelier’s principle. Consider the dissolution of silver iodide:
39.4K
Hydration of Cement
1.7K
Hydration of cement is a chemical reaction between cement particles and water. This process occurs primarily through two mechanisms: through-solution and topochemical. In the through-solution process, anhydrous compounds dissolve into their constituents, hydrates form in the solution, and then precipitate from the supersaturated solution. The topochemical process involves solid-state reactions at the cement particle surface. The through-solution process dominates the topochemical process at the...
1.7K
Soundness of Cement
679
The soundness of cement refers to the ability of cement paste to retain its volume after setting. Unsound cement can lead to expansion and structural damage due to the presence of free lime, magnesia, and calcium sulfate. Free lime hydrates very slowly, expanding and causing unsoundness, which is difficult to detect because it intercrystallizes with other compounds. Magnesia also reacts with water, forming crystals that can disrupt the cement's structure. Calcium sulfate can create...
679
Accelerators
348
Accelerators in concrete serve as admixtures to speed up the hardening process, enabling the concrete to achieve early strength faster. Although accelerators do not necessarily impact the time it takes concrete to set, they reduce this time in practice. A common accelerator is calcium chloride, which is particularly useful for hastening early strength development in cold weather or for rapid repair jobs that require quick heat generation after mixing.
The effectiveness of calcium chloride can...
The effectiveness of calcium chloride can...
348
Drying Shrinkage
476
When hardened concrete is exposed to air with a relative humidity of less than 100 percent, it begins to lose the free water within its capillaries. As this water evaporates, the water initially adsorbed onto the calcium silicate hydrates migrates towards these now empty spaces and eventually evaporates as well. Over time, as more water leaves, the volume of the concrete decreases, a phenomenon known as drying shrinkage.
A portion of this drying shrinkage can be reversed; if the concrete is...
A portion of this drying shrinkage can be reversed; if the concrete is...
476
Alkali Aggregate Reaction in Concrete
758
The alkali-aggregate reaction in concrete involves natural siliceous minerals in aggregates reacting with alkaline hydroxides derived from cement alkalis. This reaction forms an alkali-silica gel that absorbs water, swells, and increases in volume, which is confined by the surrounding cement paste, creating internal pressures that crack and disrupt the concrete. The extent of expansion and damage can be partly attributed to the alkali-silica reaction's osmotic hydraulic pressure and the...
758


