Triccas James A, Pinto Rachel, Britton Warwick J
Centenary Institute of Cancer Medicine and Cell Biology, Newtown NSW, Australia.
Res Microbiol. 2002 Jul-Aug;153(6):379-83. doi: 10.1016/s0923-2508(02)01327-x.
The green fluorescent protein (GFP) is a useful reporter for the study of gene expression and protein localisation within living cells. The stability of GFP permits its intracellular accumulation and detection, but renders it less useful for assessing transient changes in gene expression. We have developed a destabilized form of GFP for monitoring gene expression in mycobacteria. By fusing to the C-terminal end of GFP an 11 amino acid peptide encoded by the E. coli ssrA gene, we have developed a form of GFP that exhibits gradual, time-dependent degradation within the fast-growing species Mycobacterium smegmatis. This unstable variant of GFP detected transient changes in the activity of the stress-induced Mycobacterium tuberculosis sigE promoter; by contrast, unmodified GFP only detected a delayed 'switch-on' of this promoter upon exposure to acid stress. Both forms of the protein displayed equivalent stability in the slow-growing species Mycobacterium bovis bacille Calmette-Guerin (BCG), suggesting differing recognition of the ssrA-encoded peptides in slow- and fast-growing mycobacteria. This system will facilitate studies exploring dynamic changes in mycobacterial gene expression.
绿色荧光蛋白（GFP）是研究活细胞内基因表达和蛋白质定位的一种有用的报告分子。GFP的稳定性使其能够在细胞内积累并被检测到，但这也使其在评估基因表达的瞬时变化方面用处较小。我们开发了一种不稳定形式的GFP，用于监测分枝杆菌中的基因表达。通过将由大肠杆菌ssrA基因编码的11个氨基酸的肽与GFP的C末端融合，我们开发了一种在快速生长的耻垢分枝杆菌中表现出逐渐的、时间依赖性降解的GFP形式。这种不稳定的GFP变体检测到了应激诱导的结核分枝杆菌sigE启动子活性的瞬时变化；相比之下，未修饰的GFP仅在暴露于酸应激时检测到该启动子的延迟“开启”。这两种形式的蛋白质在缓慢生长的卡介苗（BCG）中显示出同等的稳定性，这表明在缓慢生长和快速生长的分枝杆菌中对ssrA编码肽的识别不同。该系统将有助于探索分枝杆菌基因表达动态变化的研究。
