RESUMO
This research focuses on the construction of an affinity purification system based on Cfa DnaE split intein. Cfa DnaE intein is an artificially constructed intein with the advantages of a fast cleavage reaction and good stability. In a previous study, a purification system that uses Cfa intein as a tag was constructed, the separation of the target protein and the tag during the purification process was completed, and the purity of the purified target protein reached 98.21%. Guided by molecular docking results, we identified flexible regions in the split intein and inserted several glycines into the protein to decrease the stability of the Cfa IC , thereby improving the regenerability of the IN media. Inserting 6 glycines between amino acids 14 and 15 of IC improved the regeneration rate of IC -GFP on the column to approximately 96%.
Assuntos
DNA Polimerase III , Inteínas , Cromatografia de Afinidade/métodos , DNA Polimerase III/metabolismo , Glicina , Inteínas/genética , Simulação de Acoplamento MolecularRESUMO
A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9-21.0 mg mL-1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.
Assuntos
Biotecnologia/métodos , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Proteínas Recombinantes/isolamento & purificação , Álcool Desidrogenase/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Ditiotreitol/química , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Inteínas , Ligantes , Domínios Proteicos , Processamento de Proteína , Proteínas Recombinantes/química , Zinco/químicaRESUMO
Oral and pharyngeal cancer is the sixth most common cancer worldwide, the 5-year survival rate has not yet increased. A key factor in rates not having improved is the lack of early detection. This study was undertaken to estimate the diagnostic accuracy of brush biopsy with DNA-image cytometry (a noninvasive method) for potentially malignant oral disorders compared with tissue biopsy pathology in China. Exfoliative cells were obtained using a cytobrush cell collector from oral mucosa of 52 subjects, followed by scalpel biopsy from the same region. Nuclear DNA contents (ploidy) were measured after Feulgen restaining, using an automated DNA image cytometer. Exfoliative cytology with DNA-image cytometry and histopathological diagnosis were performed separately at different institutions. Histological investigation was considered the gold standard. We reported that the sensitivity of DNA aneuploidy for the detection of cancer cells in potentially malignant oral disorders was 86.36 %, its specificity was 90.00 %, its positive predictive value was 86.36 %, and its negative predictive value was 90.00 %. Brush biopsy with DNA-image cytometry is a useful method for monitoring potentially malignant oral disorders.
