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长双歧杆菌NCC2705葡萄糖与乳糖代谢的比较蛋白质组学

来源:中科院微生物研究所 2008-12-13 18:01

长双歧杆菌NCC2705葡萄糖与乳糖代谢的比较蛋白质组学

何湘1 **,刘大伟1 **,孙忠科1,王芳1,姜铮1,赵红庆1,陈宣男2,黄留玉1,袁静1*

(1军事医学科学院疾病预防控制研究所,北京100071)

(2吉林大学公共卫生学院,长春 130021)

摘要:【目的】以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础,研究长双歧杆菌发酵乳糖和葡萄糖的比较蛋白质组学。【方法】采用ImageMaster 2D Elite Platnum Version 5.0 比较分析3倍以上蛋白差异点;利用MALDI-TOF进行差异蛋白鉴定, 每个蛋白质点的肽指纹图谱在长双歧杆菌NCC2705菌株的蛋白质数据库用Mascot进行检索;采用Pro-Q磷酸化试剂进行磷酸化蛋白的染色。【结果】鉴定到31个蛋白表达发生显著变化,在乳糖发酵中14 个蛋白上调17个蛋白下调。这些蛋白为亲水性酸性蛋白,它们基因的CAI值均在0.5以上,主要包括糖代谢相关蛋白、应激蛋白、转录和翻译相关蛋白,还有一些未知功能的蛋白。此外,有两个蛋白:转醛缩酶(BL0715,transaldolase,tal)L3蛋白点和丙酮酸激酶(BL0988, pyruvate kinase, pyk)G9蛋白点发生了磷酸化作用。【结论】长双歧杆菌NCC2705在乳糖中生长快于葡萄糖,它们的降解途径是相同的;转醛缩酶和丙酮酸激酶发生了翻译后修饰作用,推测转醛缩酶在43T和47S发生了磷酸化,而丙酮酸激酶在65S发生了磷酸化。

关键词:比较蛋白质组;乳糖代谢;双向电泳;Pro-Q Diamond 染色;MALDI-TOF;磷酸化蛋白

中图分类号:Q935    文献标识码:A   文章编号:0001-6209 (2008) 11-1451-08

Proteomic analysis of Bifidobacteria longum strain NCC2705 grown on lactose and glucose

Xiang He 1**, Dawei Liu 1**, Zhongke Sun1, Fang Wang 1, Zheng Jiang 1,

Hongqing Zhao 1, Xuannan Chen 2, Liuyu Huang1, Jing Yuan 1*

(1 Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China)

(2 School of Public Health, Jilin University, Changchun 130021, China)

Abstract: [Objective] Based on a proteomic reference map of the important probiotic organism Bifidobacteria longum NCC2705 constructed by our previous research, we compared the proteomic profiles of Bifidobacteria longum strain NCC2705 grown on lactose or glucose to identify the catabolic route allowing lactose fermentation. [Methods] We considered the proteins differentially expressed if their relative volume deviated more than 3-fold with ImageMaster 2D Elite version 5.0 software. Interesting spots were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and phosphorylation analysis of proteins with mobility changes by Pro-Q Diamond Stain. [Results] The identified spots represent 31 protein entries, 14 up-regulated proteins, 17 down-regulated proteins. These identified proteins, which were hydrophilic proteins and their genes with CAI value above 0.5 represented the most abundant proteins, included key stress proteins, metabolism-related proteins, and proteins related to translation. Two proteins including Tal (BL0715, transaldolase, L3) and Pyk (BL0988, pyruvate kinase, G9) exhibited clear post-translational modification. [Conclusion] Proteomic comparison of glucose- and lactose-grown cells revealed that lactose and glucose were catabolized via the same degradation pathway, and the rate of glucose assimilation was higher than that of lactose. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Pro-Q Diamond staining analysis revealed that lactose trigger Tal phosphorylation at 43 T /47 S, and inhibited Pyk phosphorylation at 65 S. These proteins were identified for the first time as bifidobacterial phosphoproteins.

Keywords: Comparative proteome; the catabolism of lactose; 2D-PAGE; Pro-Q Diamond staining; phosphoproteins

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