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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
[Cloning, expression, purification of protein kinase Cdelta and its preliminary application in drug lead compounds
1The College of Life Science, East China Normal University, Shanghai 200062, China. ys02261040@student.ecnu.edu.cn
Abstract:
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.
Insights
Researchers developed active recombinant Protein Kinase Cdelta (PKCdelta) for drug discovery. This purified and active enzyme is crucial for screening potential drug candidates targeting diabetes and cancer.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Protein Kinase Cdelta (PKCdelta) is a key enzyme in cellular signaling pathways.
- Abnormal PKCdelta activation is implicated in the pathogenesis of diabetes and various cancers.
- Targeting PKCdelta with specific inhibitors presents a promising therapeutic strategy for these diseases.
Purpose of the Study:
- To produce active recombinant mouse PKCdelta protein for further research.
- To establish a reliable method for obtaining purified and functional PKCdelta.
- To facilitate drug lead compound screening for PKCdelta inhibitors.
Main Methods:
- Designing specific primers for mouse PKCdelta gene amplification.
- Utilizing RT-PCR to obtain full-length coding cDNA.
- Cloning into a FLAG-tagged expression vector (pcDNA3.0) and sequence verification.
- Transfection into COS1 cells and stable expression selection using G418.
- Purification of FLAG-tagged PKCdelta using anti-FLAG affinity chromatography.
- Analysis of protein purity via SDS-PAGE and Western blotting.
- Enzyme activity assessment using kinase assays and inhibition studies.
Main Results:
- Successfully cloned and expressed active recombinant mouse PKCdelta in COS1 cells.
- Purified PKCdelta demonstrated high chemical and antigenic purity.
- Kinase assays confirmed the enzymatic activity of the recombinant protein.
- Inhibition studies using staurosporine validated the enzyme's responsiveness to inhibitors.
- Preliminary screening identified compounds with inhibitory effects on PKCdelta.
Conclusions:
- The successful production of active, purified recombinant PKCdelta provides a valuable tool for biochemical and pharmacological studies.
- This recombinant enzyme is suitable for high-throughput screening of potential drug candidates targeting PKCdelta.
- The findings support the development of novel therapeutics for diabetes and cancer by targeting PKCdelta.
