[目的]假單胞菌SJTE-1可高效轉(zhuǎn)化17β-雌二醇,但其代謝機(jī)制尚不清楚。本文鑒定和表征了該菌株中參與雌二醇降解與調(diào)控過(guò)程的17β-羥甾類(lèi)脫氫酶2 (17β-HSD2)和轉(zhuǎn)錄調(diào)控因子AraC。[方法]我們通過(guò)熒光定量PCR分析了17β-hsd2和araC的轉(zhuǎn)錄水平;我們?cè)诖竽c桿菌BL21(DE3)菌株中異源表達(dá)了17β-HSD2和AraC基因,并利用金屬離子親和層析法純化獲得了重組蛋白;我們體外表征了17β-HSD2的酶學(xué)性質(zhì),利用高效液相色譜鑒定了其產(chǎn)物;通過(guò)電泳遷移轉(zhuǎn)移法和DNase酶I足跡試驗(yàn),我們鑒定了重組蛋白AraC的結(jié)合能力與結(jié)合位點(diǎn)。[結(jié)果]17β-HSD2和AraC可被17β-雌二醇誘導(dǎo)表達(dá);蛋白序列比對(duì)結(jié)果表明17β-HSD2含有短鏈脫氫酶/還原酶(SDR)和β-羥甾類(lèi)脫氫酶的保守結(jié)構(gòu)與殘基。該酶以NAD+為輔助因子,在C₁₇位點(diǎn)氧化17β-雌二醇,其K_m值為0.082 mmol/L,V_max值為56.26±0.02μmol/(min·mg);5 min內(nèi)可轉(zhuǎn)化97.4%以上的雌二醇。轉(zhuǎn)錄調(diào)控因子Ar...

【文章頁(yè)數(shù)】：14 頁(yè)

【文章目錄】：
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



本文編號(hào)：3757968