Immobilization of Malarial (Plasmodium falciparum) Dihydrofolate Reductase for the Selection of Tight-Binding Inhibitors from Combinatorial Library
详细信息    查看全文
文摘
A simple procedure for selection of tight-binding inhibitorsof mutant dihydrofolate reductases from Plasmodiumfalciparum (PfDHFRs) based on preferential binding tothe enzyme immobilized on a Sepharose column has beendescribed. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to athiopropyl-Sepharose gel via S-S linkage. The amount ofimmobilized DHFRs was estimated to be 4-5 mg/g ofdried gel, and the activities of bound DHFRs werecomparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based onthe affinities of each ligand with the enzyme. Free ligandswere then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry,and the components with high binding affinity of thelibrary could thus be realized. Results could be confirmedby quantitative analysis of the bound ligands released fromthe enzyme by guanidine hydrochloride treatment.

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址:北京市海淀区学院路29号 邮编:100083

电话:办公室:(+86 10)66554848;文献借阅、咨询服务、科技查新:66554700