恶臭假单胞菌SJTE-1中氧化17β-雌二醇的17β-羟甾类脱氢酶2及其转录调控因子AraC的鉴定（英文）

发布时间：2023-03-09 07:21

　　[目的]假单胞菌SJTE-1可高效转化17β-雌二醇,但其代谢机制尚不清楚。本文鉴定和表征了该菌株中参与雌二醇降解与调控过程的17β-羟甾类脱氢酶2 (17β-HSD2)和转录调控因子AraC。[方法]我们通过荧光定量PCR分析了17β-hsd2和araC的转录水平;我们在大肠杆菌BL21(DE3)菌株中异源表达了17β-HSD2和AraC基因,并利用金属离子亲和层析法纯化获得了重组蛋白;我们体外表征了17β-HSD2的酶学性质,利用高效液相色谱鉴定了其产物;通过电泳迁移转移法和DNase酶I足迹试验,我们鉴定了重组蛋白AraC的结合能力与结合位点。[结果]17β-HSD2和AraC可被17β-雌二醇诱导表达;蛋白序列比对结果表明17β-HSD2含有短链脱氢酶/还原酶(SDR)和β-羟甾类脱氢酶的保守结构与残基。该酶以NAD+为辅助因子,在C₁₇位点氧化17β-雌二醇,其K_m值为0.082 mmol/L,V_max值为56.26±0.02μmol/(min·mg);5 min内可转化97.4%以上的雌二醇。转录调控因子Ar...

【文章页数】：14 页

【文章目录】：
1 Materials and methods
    1.1 Strains,chemicals and cultures
    1.2 Standard DNA manipulation
    1.3 Multiple sequences alignment(MSA)and homology modelling
    1.4 Reverse transcription and quantitative PCR(RT-qPCR)
    1.5 Heterogeneous expression and affinity purification of recombinant proteins
    1.6 Enzymatic assay of 17β-HSD2
    1.7 High Performance Liquid Chromatography(HPLC)detection of enzymatic products
    1.8 Green fluorescent protein(GFP)fluorescence assay
    1.9 Electrophoretic mobility shift assay(EMSA)
    1.1 0 Detection of estrogens effect on protein-DNA interaction
    1.1 1 DNase I foot-printing assay
2 Results
    2.1 Sequence alignments and structure conformation showed that 17β-HSD2 contained the conserved structure of SDR
    2.2 Protein ANI03512.1 could be induced by17β-estradiol and was a 17β-HSD
    2.3 AraC could bind to the speicific sites in the promoter region of 17β-hsd2 gene and repress the expression of 17β-HSD2



本文编号：3757968