Objective: To express and purify recombinant glutamine:fructose-6-phosphate amidotransferase(GFAT) with biological activity. Methods: Human GFA T cDNA was obtained from human liver tissue and full length of GFA Tl gene was cloned into the pET32b plasmid. Then the recombinant plasmid was transformed into Esckerichia coli Origami (DE3) and the expressed recombinant protein was purified by His-Bind column. The activity of purified recombinant protein was assayed by GDH method. Results: The prokaryotie expression vector pET32b-GFAT1 was successfully constructed. After expression and purification, the GFAT protein was obtained and its biological activity was confirmed by GDH methods. Conclusion: The recombinant human GFAT protein was obtained by prokaryotie expression system, and it shows biological activity by enzymatic method ire vitro.%目的:制备重组谷氨酰胺∶6-磷酸果糖酰胺转移酶(GFAT),检测其活性.方法:利用RT-PCR扩增人肝脏cDNA中GFA T1基因全长片段,克隆到表达载体pET32b中；在大肠杆菌Origami (DE3)中诱导表达,用镍离子螯合柱(Ni-NTA)纯化重组GFAT1；用体外酶学的方法检测GFAT的活性.结果:构建了pET32b-GFAT1质粒,经诱导表达及纯化,得到具有一定生物活性的GFAT.结论:利用原核表达系统可得到具有良好生物学活性的重组人GFAT1.
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