el-Maghrabi M R, Pilkis S J
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
Biochem Biophys Res Commun. 1991 Apr 15;176(1):137-44. doi: 10.1016/0006-291x(91)90900-r.
Rat liver fructose-1,6-bisphosphatase was expressed in Escherichia coli using a T7 RNA polymerase-transcribed expression system. Maximum yields of soluble active enzyme were obtained when the bacterial host cell, BL21(DE3), carrying the expression plasmid was grown and transcription induced in LB medium at 37 degrees C. Approximately 20mg of fructose-1,6-bisphosphatase are synthesized per liter of culture after 4hr, of which about 10mg are soluble and enzymatically active. Expressed fructose-1,6-bisphosphatase, purified to homogeneity by substrate elution from a carboxymethyl Sephadex column, was indistinguishable from that purified from rat liver in terms of subunit size and kinetic properties. The in vitro expression of fructose-1,6-bisphosphatase in an heterologous system is a necessary preliminary step for future studies on site-directed mutant enzyme forms.
利用T7 RNA聚合酶转录表达系统在大肠杆菌中表达大鼠肝脏果糖-1,6-二磷酸酶。当携带表达质粒的细菌宿主细胞BL21(DE3)在LB培养基中于37℃生长并诱导转录时，可获得可溶性活性酶的最大产量。培养4小时后，每升培养物中约合成20mg果糖-1,6-二磷酸酶，其中约10mg是可溶且具有酶活性的。通过从羧甲基葡聚糖凝胶柱上进行底物洗脱纯化至同质的表达型果糖-1,6-二磷酸酶，在亚基大小和动力学性质方面与从大鼠肝脏中纯化的酶没有区别。果糖-1,6-二磷酸酶在异源系统中的体外表达是未来对定点突变酶形式进行研究的必要初步步骤。
