RESUMO
By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase. Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater. beta-Galactosidase molecules obtained from two different crystals had indistinguishable activity distributions of 31,600 +/- 1100 and 31,800 +/- 1100 reactions min(-1) (enzyme molecule)(-1). This activity was found to be significantly different from that of the enzyme used to grow the crystals, which showed an activity distribution of 38,500 +/- 900 reactions min(-1) (enzyme molecule)(-1).
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , beta-Galactosidase/química , beta-Galactosidase/isolamento & purificação , Cristalização , Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Ativação Enzimática , Proteínas de Escherichia coli/normas , Corantes Fluorescentes/normas , Galactosídeos/normas , Oxazinas/normas , Especificidade por Substrato , beta-Galactosidase/normasRESUMO
Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.