Joint BioEnergy Institute, Emeryville, CA 94608, USA.
Proteomics. 2012 Apr;12(8):1289-99. doi: 10.1002/pmic.201100482.
As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways.
随着合成生物学的发展，其逐渐能够与商品和高价值化合物的化学转化相竞争，因此需要各种各样经过良好表征的生物部件来促进系统设计。基于选择反应监测（SRM）质谱的蛋白质定量分析补充了代谢物和转录本分析，有助于对系统进行特征描述，并优化通过工程途径的通量。通过使用 SRM 定量分析，我们检测了来自含有几种诱导型和组成型启动子的质粒表达的红色荧光蛋白（RFP），并随后评估了相同启动子驱动 8 种甲羟戊酸途径蛋白在大肠杆菌中表达的蛋白生产情况。对于每个启动子系统，操纵子中第一个基因的蛋白水平紧随 RFP 之后，但来自远离启动子的基因的蛋白产量则不太一致。其次，我们使用靶向蛋白质组学来表征酪氨酸生物合成途径蛋白在去除天然调控后的情况。这些变化预计不会对蛋白水平产生重大影响，但观察到蛋白丰度存在显著差异，这些菌株的酪氨酸产量范围从低于 1mg/L 到高于 250mg/L。总的来说，我们的结果强调了靶向蛋白质组学在确定工程系统中准确蛋白水平和微调代谢途径方面的重要性。
