Ali Rizwan, Ramadurai Sivaramakrishnan, Barry Frank, Nasheuer Heinz Peter
Systems Biology Ireland NUI Galway Ireland.
Biochemistry School of Natural Sciences and Centre for Chromosome Biology National University of Ireland Galway Ireland.
FEBS Open Bio. 2018 May 8;8(6):1043-1060. doi: 10.1002/2211-5463.12432. eCollection 2018 Jun.
The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule-based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5'-UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5'-UTR.
荧光融合蛋白（FFP）表达水平的调控是现代生物学中重组DNA技术的核心，因为蛋白质的过表达会导致生物学实验中出现假象。此外，一些显微镜技术，如荧光相关光谱法（FCS）和基于单分子的技术，对FFP的高表达水平非常敏感。为了与常用的非常强的巨细胞病毒（CMV）启动子相比降低重组蛋白的表达水平，对单纯疱疹病毒胸苷激酶（TK）基因启动子及其突变体进行了分析。构建了TK启动子的缺失突变体，并将其引入Gateway系统，用于异位表达增强型绿色荧光蛋白（eGFP）、单体樱桃红（mCherry）以及包含这些荧光蛋白的FFP。建立了两种启动子构建体TK2ST和TKTSC，它们具有适合在U2OS、HeLa CCL2、NIH 3T3和BALB/c细胞中进行FCS研究的最佳低表达水平。有趣的是，当在这四种细胞系中进行测试时，在TK基因5'-UTR内有缺失的启动子构建体显示出比缺乏该缺失的等效构建体显著更高的蛋白表达水平。这表明一个负调控元件位于TK基因的5'-UTR内。
