摘要
目的对依赖于靶点稳定性的药物亲和反应（DARTS）这一靶标发现新技术进行主要影响因素的探索,获得DARTS更适宜的实验条件。方法采用不同浓度的链霉蛋白酶（酶与蛋白质量比1∶100~1∶10 000）对Jurkat细胞提取的蛋白混合物进行室温酶解,通过聚丙烯酰胺凝胶电泳（SDS-PAGE）和考马斯亮蓝染色,观察不同浓度链霉蛋白酶对蛋白混合物酶解的程度,以确定DARTS适宜的酶解浓度范围。在此基础上,以α-酮戊二酸为研究对象,采用DARTS联合SDS-PAGE,考察不同链霉蛋白酶浓度（酶与蛋白质量比为1∶500~1∶5000）和不同酶解时长（5~30 min）对DARTS结果的影响,获得适宜的酶解条件。选用靶标已知的麦考酚酸,采用上述实验所得的最适酶解条件进行DARTS,验证其可行性。结果采用不同浓度链酶蛋白酶对蛋白混合物进行酶解时,浓度为1∶100的条件下蛋白完全被水解,酶浓度为1∶10 000对蛋白混合物无明显水解,酶浓度为1∶500~1∶5000较温和,约可水解蛋白混合物的30%~60%。链霉蛋白酶浓度为1∶1000时,α-酮戊二酸的DARTS中受保护的蛋白条带呈现效果最好;在该酶浓度条件下,适宜的酶解时长为15 min。将以上酶解条件（链霉蛋白酶浓度1∶1000、酶解15 min）运用于麦考酸的DARTS,在4~7×10~4处可见明显的保护条带,Western免疫印迹法验证结果显示,保护条带中含有麦考酚酸靶蛋白IMPDH 1。结论以α-酮戊二酸为研究对象,获得了蛋白混合物DARTS更适宜的实验条件。
Objective To explore the optimal proteolysis condition for the new technology of target discovery,drug affinity responsive target stability（DARTS）. Methods First,in order to determine the suitable pronase concentration range for DARTS,the extraction of human acute T lymphoblastic leukemia cells（Jurkat）were digested with a range of pronase concentrations（the mass ratio of pronase to protein was 1∶100 to 1∶10000）at room temperature and detected by SDS-PAGE and Coomassie brilliant blue staining. On this basis,in order to obtain the optimal proteolysis condition,we performed DARTS on α-ketoglutarate in combination with SDS-PAGE and gel staining,and observed the effect of different concentrations of pronase（1∶500-1∶5000）and different proteolysis time（5-30 min）on DARTS. The feasibility of the optimum condition was verified by using it on a target known small molecule,mycophenolic acid. Results When the protein extraction of Jurkat was hydrolyzed by a range of pronase concentrations,it was entirely hydrolysed by pronase 1∶100 while showing no significant effect under the condition of pronase 1∶10000. The effect of pronase 1∶500 to 1∶5000 on protein mixture was milder. And approximately 30%-60% of the protein was digested. The protein bands which were protected by α-ketoglutarate could be observed apparently under the conditon of pronase 1∶1000 when the proteolysis time was about 15 min.Then we performed DARTS on mycophenolic acid utilizing this condition（pronase 1∶1000,hydrolysed for 15 min）and obtained several visible protected protein bands between（4-7）×10~4. Western blotting results showed that the target protein of mycophenolic acid,IMPDH1,was contained in the protected protein bands. Conclusion The optimal proteolysis condition for DARTS on protein mixture through α-ketoglutarate is obtained.
出处
《国际药学研究杂志》
CAS
CSCD
北大核心
2016年第3期485-490,共6页
Journal of International Pharmaceutical Research
基金
国家“重大新药创制”科技重大专项资助项目(2012ZX09301003-001-006,2012ZX09301003-004-002,2013ZX09402203003)
