Abstract
Cyclin dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA sci ntillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.
Keywords: Cdk4 kinase actiity, Strep tag, yclin dependent kinases, Scintillation proximity assay, Filter binding assay, Phosphorylation, GST Rb152, Biotinylated peptide
Combinatorial Chemistry & High Throughput Screening
Title: Measurement of Cdk4 Kinase Activity Using an Affinity Peptide-Tagging Technology
Volume: 3 Issue: 1
Author(s): Jinzi J. Wu, Donna R. Yarwood, Bhabatosh Chaudhuri, Lionel Muller, Mauro Zurini and Mathew A. Sills
Affiliation:
Keywords: Cdk4 kinase actiity, Strep tag, yclin dependent kinases, Scintillation proximity assay, Filter binding assay, Phosphorylation, GST Rb152, Biotinylated peptide
Abstract: Cyclin dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA sci ntillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.
Export Options
About this article
Cite this article as:
Wu J. Jinzi, Yarwood R. Donna, Chaudhuri Bhabatosh, Muller Lionel, Zurini Mauro and Sills A. Mathew, Measurement of Cdk4 Kinase Activity Using an Affinity Peptide-Tagging Technology, Combinatorial Chemistry & High Throughput Screening 2000; 3 (1) . https://dx.doi.org/10.2174/1386207003327774
DOI https://dx.doi.org/10.2174/1386207003327774 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
Call for Papers in Thematic Issues
Artificial Intelligence Methods for Biomedical, Biochemical and Bioinformatics Problems
Recently, a large number of technologies based on artificial intelligence have been developed and applied to solve a diverse range of problems in the areas of biomedical, biochemical and bioinformatics problems. By utilizing powerful computing resources and massive amounts of data, methods based on artificial intelligence can significantly improve the ...read more
Eco-friendly Agents for Biological Control of Pathogenic Diseases
The discovery of an alternative biological approach to disease management includes work on medicinal products derived from natural sources as a starting point for the development of eco-friendly agents for these diseases and the injuries they cause, as well as reducing human contact with hazardous chemicals and their residues. We ...read more
Emerging trends in diseases mechanisms, noble drug targets and therapeutic strategies: focus on immunological and inflammatory disorders
Recently infectious and inflammatory diseases have been a key concern worldwide due to tremendous morbidity and mortality world Wide. Recent, nCOVID-9 pandemic is a good example for the emerging infectious disease outbreak. The world is facing many emerging and re-emerging diseases out breaks at present however, there is huge lack ...read more
Exploring Spectral Graph Theory in Combinatorial Chemistry
Scope of the Thematic Issue: Combinatorial chemistry involves the synthesis and analysis of a large number of diverse compounds simultaneously. Traditional methods rely on brute force experimentation, which can be time-consuming and resource-intensive. Spectral Graph Theory, a branch of mathematics dealing with the properties of graphs in relation to the ...read more
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Statin-induced Vascular Smooth Muscle Cell Apoptosis: A Possible Role in the Prevention of Restenosis?
Current Drug Targets - Cardiovascular & Hematological Disorders Is Senescence Reversible?
Current Drug Targets Antagonism by Bioactive Polyphenols Against Inflammation: A Systematic View
Inflammation & Allergy - Drug Targets (Discontinued) Hsp70 Structure, Function, Regulation and Influence on Yeast Prions
Protein & Peptide Letters Short-Chain Fatty Acid Inhibitors of Histone Deacetylases: Promising Anticancer Therapeutics?
Current Cancer Drug Targets The Stem Cell Factor Receptor/c-Kit as a Drug Target in Cancer
Current Cancer Drug Targets Mesenchymal Stem Cells in the Treatment of Amyotrophic Lateral Sclerosis
Current Stem Cell Research & Therapy NK-1 Receptor Antagonists: A New Generation of Anticancer Drugs
Mini-Reviews in Medicinal Chemistry Curcumin Potentiates The Ability of Sunitinib to Eliminate the VHL-lacking Renal Cancer Cells 786-O: Rapid Inhibition of Rb Phosphorylation as a Preamble to Cyclin D1 Inhibition
Anti-Cancer Agents in Medicinal Chemistry Advances of Phenoxazines: Synthesis, Reactivity and Their Medicinal Applications
Current Medicinal Chemistry Targeting MDM4 as a Novel Therapeutic Approach for Hematologic Malignancies
Current Cancer Drug Targets HDAC as a Therapeutic Target for Treatment of Endometrial Cancers
Current Pharmaceutical Design An Integrated and Disease-Oriented Growth Factor-Regulated Signal Transduction Network
Current Molecular Medicine IP6 in Cancer Therapy: Past, Present and Future
Current Cancer Therapy Reviews Therapeutic Potential of Natural Compounds in Lung Cancer
Current Medicinal Chemistry Therapeutic Application of Natural Medicine Monomers in Cancer Treatment
Current Medicinal Chemistry Cathepsin D as a Promising Target for the Discovery of Novel Anticancer Agents
Current Cancer Drug Targets Direct Evidence on the Immune-Mediated Spontaneous Regression of Human Cancer: An Incentive for Pharmaceutical Companies to Develop a Novel Anti-Cancer Vaccine
Current Pharmaceutical Design The Impact of Molecularly Targeted Therapies Upon the Understanding of Leukemogenesis and the Role of Hematopoietic Stem Cell Transplantation in Acute Promyelocytic Leukemia
Current Stem Cell Research & Therapy Therapeutic Transfer of DNA Encoding Adenoviral E1A
Recent Patents on Anti-Cancer Drug Discovery