[Cloning, expression, purification of protein kinase Cdelta and its preliminary application in drug lead compounds screening].
Protein kinase Cdelta (PKCdelta) is a member of protein kinase C family, which possess phospholipid-dependent serine and threonine kinase activity. PKCdelta is a potential drug target of diabetes and some cancers. The abnormal activation of PKCdelta can arouse diabetes and some cancers. Therefore the specific inhibitors of PKCdelta can be applied in the research and development of the drug candidate of these diseases. The present aim is to obtain active recombinant PKCdelta from COS1 cells. For cloning of mouse PKCdelta a pair of specific primers were designed based on the published sequence of this gene. The cDNA of full coding region was obtained by RT-PCR. The amplified cDNA was subsequently cloned into FLAG-tagged pcDNA3.0 and its sequence was confirmed by DNA sequencing analysis. FLAG-tagged pcDNA3.0-PKCdelta was transfected into COS1 cells. A cell strain which can stably express PKCdelta was obtained by G418 screening. FLAG-tagged PKCdelta in the supernant of COS1 cells extracts was absorbed by anti-FLAG resin and eluted by FLAG peptide. The purified protein appeared as a single band on both SDS-PAGE and western blotting, indicating that it was chemical and antigenic pure. By kinase assay, the recombinant PKCdelta was active. Positive inhibitor, staurosporine, was used to prove the enzyme could be greatly inhibited. Several compounds have been found to inhibit the enzyme, which indicates the preliminary application in drug lead compounds screening.
Chen
Liang
L
The College of Life Science, East China Normal University, Shanghai 200062, China. ys02261040@student.ecnu.edu.cn
Zhang
Hong-Feng
HF
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016503:Drug Delivery Systems; D004353:Drug Evaluation, Preclinical; D006801:Humans; D051379:Mice; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019311:Staurosporine / Q000494:pharmacology*
D011994:Recombinant Proteins; D051745:Protein Kinase C-delta; D019311:Staurosporine