人PDE4B2全长与截短突变体的重组表达及表征

Recombinant expression and characterization of full-length and truncated mutant of human PDE4B2

目的:比较6His-SUMO融合的人全长环核苷酸磷酸二酯酶4B2(SF-h PDE4B2)及152-528aa截短突变体(STh PDE4B2)在大肠杆菌融合表达、亲和纯化及酶学特征,以明确ST-h PDE4B2用于筛选抗炎活性h PDE4B2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-h PDE4B2和STh PDE4B2;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-h PDE4B2比活性接近SF-h PDE4B2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-h PDE4B2对(R)-Rolipram主要为高亲和力结合构象,而ST-h PDE4B2主要为低亲和力结合构象。结论:与SF-h PDE4B2相比,ST-h PDE4B2用于筛选抗炎活性h PDE4B2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 lO0-fold higher than SF-hPDEgB2;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 principally in the low-affinity state for （R）-Rolipram. Conclusion：ST-hPDE4B2 may have some advantages over SF-hPDE4B2 as the target for the screening of inhibitors as potential anti-inflammatory agents.