Overexpression and refolding of human Cyclin D3. A reliable method or not?

Autor(es)

Coelho, Fernanda ^[1] ; Pepino, Rebeka de Oliveira ^[1] ; Alencar, Diandra Pinheiro ^[1] ; Santos, Jessyka Lima ^[1] ; Canduri, Fernanda ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Sao Carlos Inst Chem, Dept Chem & Mol Phys, Av Trab Sao Carlense, 400 St, BR-55135658 Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Process Biochemistry; v. 99, p. 196-201, DEC 2020.
• Citações Web of Science: 0

Resumo

Cyclin D3 is a regulatory protein associated in a variety of human tumors. Despite several studies involving Cyclin D1 and D2, there are few articles that investigate the role of Cyclin D3. Therefore, this study aimed to produce and characterize Cyclin D3 using the Escherichia coli expression system and a protein refolding protocol. The anionic detergent SDS was used to solubilize the protein, then the solution were kept at 4 degrees C to precipitate SDS. After removing the precipitate by centrifugation, the supernatant was applied to the Ni-NTA column to purify His- tagged Cyclin D3. The recombinant protein shown to be refolded in a denaturation study by fluorescence spectroscopy at increasing concentrations of guanidine hydrochloride. The protein secondary structure, evaluated by circular dichroism, is composed of 39 % alpha helix and 15 % beta-strands. In addition, the study aimed to validate the refolding protocol used to obtain Cyclin D3 from E. coli inclusion bodies. (AU)

• Processo FAPESP: 16/25893-6 - Estudo das proteinas quinases CDK8 e CDK10 a partir da expressão heteróloga em E. coli
• Beneficiário: Fernanda Canduri
• Modalidade de apoio: Auxílio à Pesquisa - Regular