Gaur Deepika, Wohlever Matthew L
Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Protein Sci. 2025 Apr;34(4):e70106. doi: 10.1002/pro.70106.
Expression and purification of recombinant proteins in Escherichia coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time-consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but they are not optimized for protein biochemistry and are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a vector series with nine different N-terminal tags and no C-terminal cloning scar. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.
在大肠杆菌中表达和纯化重组蛋白是生物化学和分子生物学的一项基础技术。表达优化需要测试不同的溶解性标签、亲和纯化技术和位点特异性蛋白酶的组合。由于这些特性分布在不同的载体系列中，并且需要不同效率的克隆策略，这种优化既费力又耗时。基于金门系统的模块化克隆试剂盒虽然存在，但它们并非针对蛋白质生物化学进行优化，并且对于许多应用（如本科研究或蛋白质纯化特性的简单筛选）来说过于复杂。理想的解决方案是让单基因合成产物或PCR产物与一系列预先组装好的金门载体兼容，这些载体在N端或C端含有广泛的纯化特性。据我们所知，不存在这样的系统。为满足这一未被满足的需求，我们对pET28b载体进行了金门改造，并开发了一套包含21种载体的系统，这些载体具有不同组合的纯化标签、溶解性结构域、可视化/标记标签和蛋白酶位点。我们还开发了一个具有九个不同N端标签且无C端克隆疤痕的载体系列。该系统是模块化的，允许用户使用他们喜欢的特性组合轻松定制载体。为了便于对克隆载体进行可视化筛选，我们优化了反向链中荧光蛋白mScarlet3的组成型表达，从而在成功克隆后实现从红色到白色的颜色变化。对模型蛋白sfGFP的测试表明，该系统具有易于可视化筛选、高效克隆和强大的蛋白表达能力。这些载体为大肠杆菌中的蛋白质工程和功能研究提供了通用的高通量解决方案。
