Purification and characterization of a recombinant pea cytoplasmic fructose-1,6-bisphosphatase

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• 作者：Jang ; Hye-Kyung ; Lee ; Sang-Won ; Lee ; Youn-Hyung ; Hahn ; Tae-Ryong
• 关键词：Cytoplasmic FBPase ; Overexpression ; Recombinant FBPase
• 刊名：Protein Expression and Purification
• 出版年：2003
• 出版时间：March, 2003
• 年：2003
• 卷：28
• 期：1
• 页码：42-48
• 全文大小：230 K

文摘

Full-length cDNA encoding pea cytoplasmic fructose-1,6-bisphosphatase (cyFBPase) was cloned from a pea cDNA library. The cloned cDNA was introduced into the Escherichia coli expression vector pET-15b. The recombinant cyFBPase was expressed in E. coli BL21 (DE3) cells in a soluble form and purified to homogeneity by Ni⁺–NTA affinity chromatography. The identity of the recombinant cyFBPase was confirmed by SDS–PAGE and immunoblot analysis using a polyclonal anti-His tag antibody. The recombinant cyFBPase was active at neutral pH ranges (6.6–9.0) and thermostable as other cyFBPases. The activation energy (E_a) and Arrhenius frequency factor were 17.4kcal/mol and 2.6×10¹²/s, respectively. The K_M and V_max values of the recombinant enzyme were calculated as 10.47μM and 109μmol/min, respectively. In case of removal of histidine tag, the K_M value was calculated as 5.03μM. The recombinant enzyme was non-competitively and competitively inhibited by AMP and fructose-2,6-bisphosphate, respectively.