重组人尿苷二磷酸葡糖醛酸转移酶2B7三个功能性突变体的构建、表达及活性比较

目的构建人尿苷二磷酸葡糖醛酸转移酶(UGT)2B7重组酶的3个功能性突变体UGT2B7*71S(A71S,211G>T),UGT2B7*2(H268Y,802C>T)和UGT2B7*5(D398N,1192G>A),以进一步研究该酶野生型和其突变体在对底物作用过程中的功能差异。方法应用细菌/杆状病毒系统,将构建的pFastBac-UGT2B7*71S,pFastBac-UGT2B7*2和pFastBac-UGT2B7*5重组质粒转化E.coli DH10Bac大肠杆菌,通过转座作用获得各自的重组粘粒(bac-mid),然后将其转染草地夜蛾(Sf)9细胞后,产生重组杆状病毒。这些病毒再感染Sf9细胞,即可获得野生型UGT2B7*1及其突变体的重组酶。野生型及突变体酶的活性以7-羟基-4-三氟甲基香豆素(7-HFC)为底物,用荧光法测定并分析比较。结果利用杆状病毒/昆虫细胞系统,成功地构建人UGT2B7重组酶的3个功能性突变体。UGT2B7*1对7-HFC的Km值为(0.331±0.018)mmol·L-1,Vmax值为(2.14±0.04)μmol·min-1·g-1蛋白;UGT2B7*71S对7-HFC的Km值为(0.260±0.026)mmol·L-1,Vmax值为(1.36±0.05)μmol·min-1·g-1蛋白;UGT2B7*2对7-HFC的Km值为(0.53±0.06)mmol·L-1,Vmax值为(9.5±0.5)μmol·min-1·g-1蛋白;UGT2B7*5对7-HFC的Km值为(0.59±0.05)mmol·L-1,Vmax值为(7.52±0.28)μmol·min-1·g-1蛋白。结论应用细菌/杆状病毒系统,成功构建了UGT2B7的3个功能性突变体,这些突变体可进一步用于对其他底物的代谢活性比较。

Construction, expression and activities of three active variants of human uridine diphosphate glucuronic acid transferase 2B7

AIM To construct three active variants of the recombinant human uridine diphosphate glucuronic acid transferase(UGT) 2B7*1, such as UGT2B7*71S(A71S,211G>T)，UGT2B7*2(H268Y,802C>T) and UGT2B7*5(D398N,1192G>A)，and study their differences between wild type and variants over substrates. METHODS According to baculovirus expression system, recombinant pFastBac-UGT2B7*71S, pFastBac-UGT2B7*2 and pFastBac-UGT2B7*5 were transformed into E.coli DH10Bac, respectively. After transposition, the respective recombinant bacmid was isolated and then used to transfect Spodoptera frugiperda 9 (Sf9) cells to generate recombinant viruses which infect Sf9 cells to express target protein. The activity of the recombinant human UGT2B7*1, UGT2B7*71S, UGT2B7*2 and UGT2B7*5 was assayed by fluorometry with 7-hydroxy-4-(trifluoromethyl)coumarin (7-HFC) as substrate. RESULTS UGT2B7*71S, UGT2B7*2 and UGT2B7*5 were constructed by using baculovirus expression system. With respect to 7-HFC, the values of K_m and V_max of wild type were (0.331±0.018)mmol·L^-1 and (2.14±0.04)μmol·min^-1·g^-1 protein, the values of K_m and V_max of UGT2B7*71S were(0.260±0.026)mmol·L^-1 and (1.36±0.05)μmol·min^-1·g^-1 protein, the values of K_m and V_max of UGT2B7*2 were (0.53±0.06)mmol·L^-1 and (9.5±0.5)μmol·min^-1·g^-1 protein, and the values of K_m and V_max of UGT2B7*5 were (0.59±0.05)mmol·L^-1 and (7.52±0.28)μmol·min^-1·g^-1 protein. CONCLUSION Three active variants of UGT2B7*1 are constructed and they can be used to study the different activation on drugs and xenobiotics.