RESUMO
S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase (SUMT) is a novel red fluorescence indicator. However, the production of SUMT in Escherichia coli is restricted by its relatively low solubility, and little is known about the red fluorescent materials that are associate with SUMT. Two individual SUMT mutations, L166A and L88R/L89G double mutant were produced by site-directed mutagenesis. Both mutants were overexpressed in E. coli and purified by Ni-NTA chromatography. The reddish mixtures isolated from the purified L88R/L89G double mutant were analyzed by UV-visible spectra scanning and mass analysis(MS). The L88R/L89G double mutant has enzymatic activity in vivo, whereas L166A mutant loses the activity. Trimethylpyrrocorphin is identified as the main constituent in the isolated pigments. The purified L88R/L89G mutant increases protein solubility, which is applied potentially as the fluorescent indicator denoting the solubility of protein fusion partner.
Assuntos
Substituição de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Genética , Fluorescência , Espectrometria de Massas , Metiltransferases , Química , Genética , Metabolismo , Peso Molecular , Mutagênese Sítio-Dirigida , Métodos , Mutação , Pigmentos Biológicos , Química , Metabolismo , Proteínas de Plantas , Química , Genética , Metabolismo , Proteínas Recombinantes , Química , Metabolismo , Solubilidade , Espectrofotometria Ultravioleta , Zea mays , GenéticaRESUMO
<p><b>BACKGROUND</b>Type 1 diabetes has been recognized as an organ specific autoimmune disease owing to the immune destruction of pancreatic islet beta cells in genetically susceptible individuals. In both human and rodent models of type 1 diabetes, such as nonobese diabetic (NOD) mice, biobreeding rats, the disease has a distinct stage characterized by immune cells infiltrating in the pancreas (insulitis). The major populations of infiltrating cells are macrophages and T lymphocytes. Therefore, immune cell infiltration of pancreatic islets may be a crucial step in the pathogenesis of type 1 diabetes. Monocyte chemoattractant protein-1 can specifically attract monocytes in vivo. Interferon induced protein-10 has chemoattractant effects on the activated lymphocytes. In this study, we analysed the expression of monocyte chemoattractant protein-1 in the pancreas of mice and interferon inducible protein-10 mRNA in the pancreas of NOD mice, and discussed their possible role in the pathogenesis of type 1 diabetes.</p><p><b>METHODS</b>The immunohistochemical method and immunoelectronmicroscopy were used to evaluate the expression of monocyte chemoattractant protein-1 in the pancreas of NOD mice and BALB/c mice. RT-PCR was used to evaluate the expression of monocyte chemoattractant protein-1 and interferon inducible protein mRNA in NOD mice.</p><p><b>RESULTS</b>Monocyte chemoattractant protein-1 was positive in the pancreas of NOD mice, whereas negative in the pancreas of BALB/C mice. RT-PCR showed that monocyte chemoattractant protein-1 and interferon inducible protein-10 mRNA could be found in the pancreas of NOD mice. Immunoelectronmicroscopy demonstrated that monocyte chemoattractant protein-1 was produced by beta cells and stored in the cytoplasm of the cells.</p><p><b>CONCLUSIONS</b>Pancreatic islet beta cells produce monocyte chemoattractantprotein-1 in NOD mice. Monocyte chemoattractant protein-1 may play an important part in the pathogenesis of type 1 diabetes by attracting monocytes/macrophages to infiltrate pancreatic islets.</p>