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学科主题: 生命有机化学
题名: Overexpression in escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach
其他题名: 菠菜叶绿体三糖磷酸酯异构化酶在大肠杆菌中高表达及其性能测定
作者: Tang GL(唐功利) ; Wang YF(王燕芳) ; Bao JS(鲍建绍) ; Chen HB(陈海宝)
通讯作者: 陈海宝
刊名: Protein Expr. Purif.
发表日期: 1999
卷: 16, 期:3, 页:432-439
收录类别: SCI
部门归属: 中国科学院上海有机化学研究所生命有机化学实验室
英文摘要: An important calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the Pl expression vector in escherichia coli. An even higher level expression, up to 36% of the total protein, was achevd by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence. Computer modeling revealed that the moderate change in the standard free energy (5'△G^o) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression. The overexpressed spinach cpTPI was solubel and fully active and was abel to be purified beyond 95% purity by DEAE-Sepharose and sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria. With D-glyceraldehyde 3-phosphate as substrate, Km(D-3-P) is 0.68 mM, V max (G-3-P) is 3.16×10^4 μmol/min.mg, and Kcat (G-3-P0 is 4.51×10^3/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04×10^3 μmol/min.mg, and 1.16 ×10^2/s, are 7.27 mM, 1.04×10^3 μmol/min.mg, and 1.16 ×10^2/s, respectively.
语种: 英语
相关网址: 查看原文
WOS记录号: WOS:000081814900009
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内容类型: 期刊论文
URI标识: http://ir.sioc.ac.cn/handle/331003/25741
Appears in Collections:生命有机化学国家重点实验室_期刊论文

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Recommended Citation:
Tang GL,Wang YF,Bao JS,et al. Overexpression in escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach[J]. Protein Expr. Purif.,1999,16(3):432-439.
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