人乳铁蛋白基因在昆虫细胞中的表达  

Overexpression, Purification and Characterization of Thermostable Catechol 2,3 dioxygenase

作　　者：张大兵[1,2] 姜育蕾 吴祥甫[1,2] 洪孟民 

机构地区：[1]中国科学院上海植物生理研究所 [2]中国科学院上海生物化学研究所

出　　处：《生物化学与生物物理学报》1998年第6期575-578,共4页

摘　　要：将人乳铁蛋白ｃＤＮＡ（ｈＬＦｃ）克隆在质粒ｐＢａｃＰＡＫ８的ＢａｍＨＩ和ＳａｃＩ位点，构建成转移质粒ｐ８ｈＬＦｃ。该质粒ＤＮＡ与ＡｃＮＰＶＢａｃＰＡＫ６病毒株基因组ＤＮＡ经共转染草地夜峨（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）培养细胞Ｓｆ－２１，在培养的贴壁细胞中挑出空斑，经过３轮纯化，结合斑点杂交筛选，获得重组病毒。用蛋白质免疫印迹法检测到在重组病毒感染的Ｓｆ－２１细胞中存在乳铁蛋白基因的表达产物。酶联免疫检测结果表明：重组人乳铁蛋白在感染后细胞的可溶性蛋白质中含量为２％，在细胞培养上清液中含量为９．５ｍｇ／Ｌ。经亲和层析纯化后的重组蛋白质在ＳＤＳ－ＰＡＧＥ和人乳铁蛋白标准品的电泳迁移率基本一致。紫外光Ａ２８０与可见光Ａ４６５的吸收比值测定表明重组人乳铁蛋白具有结合铁离子的能力。Catechol 2,3 dioxygenase(EC 1.13.11.2)from Bacillus stearothermophilus has been shown to be highly thermostable. In order to obtain sufficient enzyme for its characterization, and to analyze the molecular basis of its intrinsic stability, the gene coding for this enzyme was sub cloned and over expressed in Escherichia coli . After heat denaturation, ammonium sulfate fractionation, DEAE 52 ion exchange chromatography and phenyl Sepharose CL 4B hydrophobic chromatography, the enzyme was purified to homogeneity and the yield was 16%. The enzyme is a homotetramer. The molecular weight of each subunit is 36.4 kD as determined by SDS PAGE. Using the tyrosine difference spectrum method, the extinction coefficient of the enzyme at 279.2 nm is estimated as 89 (mmol/L) -1 ·cm -1 , the optimum temperature and pH of the enzyme is 60 ℃ and 8.0, respectively. The K m for catechol of the enzyme is 1.24×10 -5 mol/L, K cat is 24 000 min -1 . Acetone competitively inhibited the enzyme activity to catechol, with an inhibition constant K i of about 165 mmol/L. The thermostability of the enzyme is reflected by its unaltered catalytic activity over 1 h at 65 ℃. Irreversible thermal denaturation becomes significant between 70—80 ℃. The pH stability of the enzyme and its resistance toward chemical denaturation such as urea and SDS confirm the intrinsic stability of the enzyme. It was also studied that the effect of different pHs on the molecular structure of the enzyme, using circular dichroism spectra and intrinsic fluorescence analysis.

关 键 词：人乳铁蛋白cDNA 重组杆状病毒 基因表达 

分 类 号：Q78[生物学—分子生物学]