RESUMEN
The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.
Asunto(s)
Cromatografía de Afinidad/métodos , Disulfuros/química , Resinas Sintéticas/química , alfa-Fetoproteínas/química , alfa-Fetoproteínas/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Metales/química , Unión Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Human alpha-fetoprotein (hAFP) is an oncofetal protein which is a common cancer marker. Conjugates of native hAFP with different cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The hAFP interacts with its receptor (AFPR) on the surface of cancer cells via its C-terminal domain. The aim of this work was to develop a highly efficient expression system in Escherichia coli and efficient refolding procedure for the recombinant C-terminal fragment of hAFP (rAFP-Cterm) and to characterize its functional properties. C-terminal fragment of hAFP (rAFP-Cterm) comprising amino acids from 404 to 609 was expressed in E. coli BL21 (DE3) strain with high yield. High efficient purification and refolding procedures were developed giving yield of refolded protein about 80% with purity about 95%. The refolded rAFP-Cterm bound specifically with cancer cells carrying AFPR and was accumulated by them with the same efficiency as native hAFP. This rAFP-Cterm can be used as a vehicle for the targeted delivery of drugs to cancer cells.
Asunto(s)
Fragmentos de Péptidos/biosíntesis , alfa-Fetoproteínas/biosíntesis , Línea Celular Tumoral , Cromatografía en Gel , Cromatografía de Fase Inversa , Dicroismo Circular , Sistemas de Liberación de Medicamentos , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , alfa-Fetoproteínas/química , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/aislamiento & purificaciónRESUMEN
The specific receptor of alpha-fetoprotein (AFP) is a universal tumor marker, being expressed on the surface of many tumor cells, but not in normal human tissues. AFP enters the cell by receptor-mediated endocytosis; its receptor-binding site is hypothetically localized in the third domain of AFP. A recombinant C-terminal AFP fragment, which contains all the third and a part of the second domains of hAFP, was produced. This AFP fragment was bound specifically to the AFP receptor on the surface of tumor cells and was accumulated by them with the same efficiency as the full-size hAFP. Similar to hAFP, the recombinant C-terminal fragment inhibited the estradiol-induced growth of hormone-dependent MCF-7 cells in vitro. Hence, the recombinant C-terminal AFP fragment can be used as a protein vector for the targeted delivery of cytostatic drugs to tumor cells.
Asunto(s)
Portadores de Fármacos/farmacología , alfa-Fetoproteínas/farmacología , Antineoplásicos/administración & dosificación , Bacterias/efectos de los fármacos , Bacterias/genética , Línea Celular Tumoral , ADN Complementario/biosíntesis , ADN Complementario/genética , Escherichia coli/metabolismo , Estradiol/farmacología , Femenino , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Microscopía Fluorescente , Pliegue de Proteína , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/farmacología , alfa-Fetoproteínas/genéticaRESUMEN
Glioblastomas belong to the most aggressive human cancers with short survival times. Due to the blood-brain barrier, they are mostly inaccessible to traditional chemotherapy. We have recently shown that doxorubicin bound to polysorbate-coated nanoparticles crossed the intact blood-brain barrier, thus reaching therapeutic concentrations in the brain. Here, we investigated the therapeutic potential of this formulation of doxorubicin in vivo using an animal model created by implantation of 101/8 glioblastoma tumor in rat brains. Groups of 5-8 glioblastoma-bearing rats (total n = 151) were subjected to 3 cycles of 1.5-2.5 mg/kg body weight of doxorubicin in different formulations, including doxorubicin bound to polysorbate-coated nanoparticles. The animals were analyzed for survival (% median increase of survival time, Kaplan-Meier). Preliminary histology including immunocytochemistry (glial fibrillary acidic protein, ezrin, proliferation and apoptosis) was also performed. Rats treated with doxorubicin bound to polysorbate-coated nanoparticles had significantly higher survival times compared with all other groups. Over 20% of the animals in this group showed a long-term remission. Preliminary histology confirmed lower tumor sizes and lower values for proliferation and apoptosis in this group. All groups of animals treated with polysorbate-containing formulations also had a slight inflammatory reaction to the tumor. There was no indication of neurotoxicity. Additionally, binding to nanoparticles may reduce the systemic toxicity of doxorubicin. This study showed that therapy with doxorubicin bound to nanoparticles offers a therapeutic potential for the treatment of human glioblastoma.