人PDE4B2全长与截短突变体的重组表达及表征

truncated mutant of human PDE4B2

摘要:

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。

Abstract:

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.