| Autor: |
Suzuki MF; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Almeida LA; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Pomin SA; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Silva FD; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Freire RP; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Oliveira JE; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Affonso R; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Soares CRJ; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil., Bartolini P; Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, Avenida Prof. Lineu Prestes 2242, São Paulo, SP, 05508-000, Brazil. pbartoli@ipen.br. |
| Abstrakt: |
The human prolactin antagonist Δ 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 Δ 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 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/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. |
