Ortega Claudia, Prieto Daniel, Abreu Cecilia, Oppezzo Pablo, Correa Agustín
Recombinant Protein Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay.
Research Laboratory on Chronic Lymphocytic Leukemia, Institut Pasteur de Montevideo, Montevideo, Uruguay.
Front Microbiol. 2018 Jun 27;9:1384. doi: 10.3389/fmicb.2018.01384. eCollection 2018.
Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.
重组蛋白表达已成为基础研究和应用研究中一项极具价值的工具。该领域积累的知识使得数以千计的蛋白质靶点能够以可溶、纯净且均一的状态表达，这对于生化分析和结构分析至关重要。在过去几十年里取得了很大进展，通过评估不同参数（如宿主、菌株、融合伴侣或启动子强度等），一些具有挑战性的蛋白质得以以可溶方式表达。在这方面，我们之前开发了一套载体，通过简单高效的克隆策略来评估不同的启动子和溶解性增强蛋白。尽管如此，许多靶点的正确表达仍然难以实现，例如需要添加复杂的翻译后修饰和/或通过特定的细胞区室。为了克服在单一亚细胞定位和单一宿主类型研究中发现的局限性，我们在此扩展了之前开发的载体套件，以评估不同细胞区室和细胞宿主中的重组蛋白表达。此外，这些载体还允许评估替代纯化策略以提高目标蛋白产量。
