A rapid method for the purification of wild-type and V804M mutant ret catalytic domain: A tool to study thyroid cancer.
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.
Department of Experimental Oncology, National Cancer Institute, Milan, Italy. elisa.sala@unimib.it
Elisa
E
Sala
Luca
L
Mologni
Sara
S
Cazzaniga
Elena
E
Papinutto
Carlo
C
Gambacorti-Passerini
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000627:therapeutic use; D006801:Humans; D010880:Piperidines / Q000627:therapeutic use; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011720:Pyrazoles / Q000627:therapeutic use; D011743:Pyrimidines / Q000627:therapeutic use; D011799:Quinazolines / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology; D013964:Thyroid Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; D010880:Piperidines; D011720:Pyrazoles; D011743:Pyrimidines; D011799:Quinazolines; D011994:Recombinant Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C452423:vandetanib