Periplasmic synthesis and purification of the human prolactin antagonist Delta(1-11)-G129R-hPRL

Autor(es)

Suzuki, Miriam F. ^[1] ; Almeida, Larissa A. ^[1] ; Pomin, Stephanie A. ^[1] ; Silva, Felipe D. ^[1] ; Freire, Renan P. ^[1] ; Oliveira, Joao E. ^[1] ; Affonso, Regina ^[1] ; Soares, Carlos R. J. ^[1] ; Bartolini, Paolo ^[1]

Afiliação do(s) autor(es)

^[1] IPEN CNEN SP, Inst Pesquisas Energet & Nucl, Ctr Biotechnol, Ave Prof Lineu Prestes 2242, BR-05508000 Sao Paulo, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: AMB EXPRESS; v. 11, n. 1 APR 27 2021.
• Citações Web of Science: 0

Resumo

The human prolactin antagonist Delta(1-11)-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Delta(1-11)-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 degrees C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 +/- 0.015 mu g/mL/A(600) at a final optical density of 3.43 +/- 0.13 A(600). Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF-MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine. (AU)

• Processo FAPESP: 12/24345-4 - Avaliação do potencial de antagonistas de prolactina para o tratamento da obesidade e do Diabetes mellitus 2
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/50332-0 - Capacitação científica, tecnológica e em infraestrutura em radiofármacos, radiações e empreendedorismo a serviço da saúde (PDIp)
• Beneficiário: Marcelo Linardi
• Modalidade de apoio: Auxílio à Pesquisa - Programa Modernização de Institutos Estaduais de Pesquisa
• Processo FAPESP: 15/26058-0 - Estudo do perfil de N-glicosilação e dos sítios de ocupação em várias preparações de tireotropina humana (hTSH) hipofisária e recombinante
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular