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重庆医科大学检验医学院,重庆,400016
纸质出版日期:2018,
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张祥, 何姝, 胡小蕾, 等. 人PDE4B2全长与截短突变体的重组表达及表征[J]. 重庆医科大学学报, 2018,43(1):93.
Zhang Xiang, He Shu, Hu Xiaolei, et al. truncated mutant of human PDE4B2[J]. Journal of Chongqing Medical University, 2018,43(1):93.
目的:比较6His-SUMO融合的人全长环核苷酸磷酸二酯酶4B2(SF-hPDE4B2)及152-528aa截短突变体(ST-hPDE4B2)在大肠杆菌融合表达、亲和纯化及酶学特征,以明确ST-hPDE4B2用于筛选抗炎活性hPDE4B2抑制剂的优势。方法:分别将6His-SUMO融合到人全长PDE4B2基因编码区的N端和152-528aa截短突变体N端得到SF-hPDE4B2和ST-hPDE4B2;大肠杆菌BL21(DE3)诱导表达,Ni2+-NTA亲和层析纯化,用偶联碱性磷酸酶的孔雀绿定磷法测定酶活性。结果:纯化后ST-hPDE4B2比活性接近SF-hPDE4B2的40倍,活性收率超过100倍;SDS-PAGE和6His标签免疫印迹发现纯化后2种融合蛋白都有多种带6His标签的降解片段;测定咯利普兰[(R)-Rolipram]和罂粟碱的抑制常数表明,SF-hPDE4B2对(R)-Rolipram主要为高亲和力结合构象,而ST-hPDE4B2主要为低亲和力结合构象。结论:与SF-hPDE4B2相比,ST-hPDE4B2用于筛选抗炎活性hPDE4B2抑制剂可能更有优势。
Objective:To compare the expression,affinity purification and enzyme kinetics against representative inhibitors of full-length human phsphodiesterase 4B2 fused with 6His-SUMO on N-terminus(SF-hPDE4B2) and its 152-528aa truncate fused with 6His-SUMO on N-terminus(ST-hPDE4B2),to reveal the superiority of ST-hPDE4B2 over SF-hPDE4B2 as the target for high-throughput screen of hPDE4B2 inhibitors as potential anti-inflammatory agents. Methods:The two fusion proteins were expressed in Escherichia coli BL 21(DE3) and purified through Ni2+-NTA;phsphodiesterase activities were measured via alkaline phosphatase-coupled malachite green assay of phosphate in high-throughput mode. Results:After affinity purification,ST-hPDE4B2 exhibited spe-cific activity nearly 40-fold higher and activity yield over 100-fold higher than SF-hPDE4B2;SDS-PAGE and Western blot of 6His tag supported that there were a lot of polypeptides degraded in both of the purified fusion proteins. Inhibition constants of (R)-Rolipram and papaverine indicated that SF-hPDE4B2 mainly existed in the high-affinity state while ST-hPDE4B2 appeared princi-pally in the low-affinity state for (R)-Rolipram. Conclusion:ST-hPDE4B2 may have some advantages over SF-hPDE4B2 as the tar-get for the screening of inhibitors as potential anti-inflammatory agents.
环核苷酸磷酸二酯酶4B2重组表达孔雀绿定磷法酶抑制剂
cyclic nucleotide phosphodiesterase 4B2fusion expressionalkaline phosphatase-coupled malachite green assay of phos-phaterolipram
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