RESUMO
Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti-LDL(-) scFv, previously obtained from a hybridoma secreting an anti-LDL(-) monoclonal antibody, was inserted into the bacterial vector pET-28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP-scFv was assessed by ELISA, which determined its affinity for LDL(-) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(-). In conclusion, our data suggest that the anti-LDL(-) GFP-scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis.
Assuntos
Biomarcadores/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Aterosclerose/metabolismo , Linhagem Celular Tumoral , Endocitose , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/farmacocinética , Camundongos , Imagem Molecular/métodos , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacocinéticaRESUMO
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
Assuntos
Proteínas de Transporte/farmacocinética , Membrana Celular/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Proteínas Estruturais Virais/farmacocinética , Animais , Western Blotting , Polpa Dentária/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Estruturais Virais/genéticaRESUMO
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.