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Updated: Aug 11, 2026
Spontaneous Murine Model of Anaplastic Thyroid Cancer
Published on: February 3, 2023
A rapid method for the purification of wild-type and V804M mutant ret catalytic domain: A tool to study thyroid
Elisa Sala1, Luca Mologni, Sara Cazzaniga
1Department of Experimental Oncology, National Cancer Institute, Milan, Italy. elisa.sala@unimib.it
Abstract:
RET (rearranged during transfection) is a transmembrane tyrosine kinase and acts as co-receptor of glial-derived neurotrophic factor (GDNF) family neurothrofic factors in complex with GFRalpha family proteins; RET is important for development of enteric nervous system and renal organogenesis during embryonal life. Alterations in Ret gene are related to several neoplasias: point mutations are identified in medullary thyroid carcinoma (MTC) and multiple endocrine neoplasias 2A and B (MEN2A and B), while translocations and chromosomal inversions cause papillary thyroid carcinoma (PTC). We expressed recombinant RET kinase domain (rRET) containing the active site, the ATP binding pocket, and the activation loop with regulatory activity, with the Baculovirus expression system. RET was purified by a two-step procedure consisting of an anion exchange chromatography followed by nickel affinity chromatography. Moreover a biochemical characterization of the recombinant product was performed in order to verify its activity (by ELISA) and physical state (dynamic light scattering). We used rRET to validate an ELISA-based kinase assay, by testing inhibitors reported in literature such as PP1 and PP2. This method represents an easy system to screen potential inhibitors found by computational methods. We also produced V804M mutants to identify inhibitors that can overcome resistance to PP1 and ZD6474. The catalytic domain of RET can be used also for X-ray diffraction to obtain information about the three-dimensional structure, necessary for a rational design of selective inhibitors: it represents an important tool to understand the molecular mechanisms causing thyroid cancer and to care it.
Insights
Recombinant RET kinase domain was produced and biochemically characterized. This recombinant RET (rRET) enables the development of novel kinase assays for screening thyroid cancer inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The RET (rearranged during transfection) proto-oncogene encodes a transmembrane tyrosine kinase crucial for nervous system and kidney development.
- RET alterations, including mutations and translocations, are implicated in various cancers, particularly thyroid carcinomas (MTC, PTC) and multiple endocrine neoplasias (MEN2A, MEN2B).
Purpose of the Study:
- To express and purify the recombinant RET kinase domain (rRET) for biochemical characterization and assay development.
- To establish a robust ELISA-based kinase assay for screening potential RET inhibitors.
- To generate RET mutants (V804M) to study inhibitor resistance and facilitate rational drug design.
Main Methods:
- Baculovirus expression system for recombinant RET kinase domain production.
- Two-step purification involving anion exchange and nickel affinity chromatography.
- Biochemical characterization using ELISA for activity and dynamic light scattering for physical state.
- Validation of an ELISA-based kinase assay with known inhibitors (PP1, PP2) and generation of V804M mutants.
Main Results:
- Successful expression and purification of active rRET kinase domain.
- Establishment and validation of an ELISA-based assay for RET kinase activity.
- Demonstrated utility of rRET in screening known inhibitors and identifying resistance-overcoming strategies.
- rRET provides a foundation for structural studies (X-ray diffraction) for rational inhibitor design.
Conclusions:
- The recombinant RET kinase domain is a valuable tool for biochemical and structural studies.
- The developed ELISA-based assay facilitates high-throughput screening of potential RET inhibitors.
- This research aids in understanding thyroid cancer molecular mechanisms and developing targeted therapies.
