利用非天然氨基酸代谢掺入法检测新生成蛋白

Detection of Newly Synthesized Proteins via Metabolic Incorporation of Non-Natural Amino Acid

以脂多糖(Lipopolysaccharide,LPS)刺激Raw264.7细胞产生肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)为模型体系,建立了非天然氨基酸代谢掺入法检测特定新生成蛋白的方法。通过代谢掺入的方法,使细胞中的蛋白质,特别是新生成的蛋白质一级结构中掺入非天然氨基酸,即带有叠氮基团的甲硫氨酸类似物(Azidohomoalanine,AHA)。考察了在不同浓度的LPS和FBS,以及不同的刺激时间等条件下,LPS刺激Raw264.7细胞产生TNF-α的实验参数,确定了最优的实验条件为:在含有1%胎牛血清的无甲硫氨酸(Met)的DMEM培养基中,分别在不加LPS和加10 ng/m L的LPS条件下刺激Raw264.7细胞4 h,在刺激细胞的同时掺入AHA。利用Cu+催化的叠氮基团与带有生物素(Biotin)标签的炔烃基团的环加成反应,使蛋白质标记上Biotin标签。利用吸附在固相载体上的抗体特异性捕获TNF-α分子,再用耦联辣根过氧化物酶(Horseradish peroxidase,HRP)的链霉亲和素(Streptavidin)对TNF-α分子上的Biotin进行识别,实现对特定的新生成蛋白质(TNF-α)进行检测。本方法为检测特定微量新生成蛋白、表征特定蛋白质的周转等研究提供了新的思路法。

A method for detection of newly synthesized protein via metabolic incorporation of non-natural amino acid was developed,in which Raw264.7 cells were treated by lipopolysaccharide(LPS)to generate tumor necrosis factor alpha(TNF-α).Experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS stimulated TNF-αfrom Raw264.7 cells.The optimal experimental conditions were determined as follows.Raw264.7 cells were cultured in Met-free DMEM containing 1%fetal bovine serum(FBS),and then treated for 4 h with LPS at concentration of 10 ng/mL in presence of AHA.The biotin tags were introduced into proteins containing azidohomoalanine(AHA)by copper-catalyzed alkyne-azidecycloaddition(CuAAC).Then TNF-αwas specifically captured by the antibody adsorbed on the solid support,on which the biotin tag could be detected by streptavidin coupled with horseradish peroxidase(HRP).This study provided a method for the detection of a specific newly synthesized protein,and characterization of specific protein turnover.