RESUMO
To examine the relationship between folding and aggregation in the periplasm of Escherichia coli, we have analysed the cellular fates of exported proteins fused to either the wild-type maltose-binding protein (MalE) or the aggregation-prone variant MalE31. The propensity of fusion proteins to aggregate in the periplasm was determined by the intrinsic folding characteristics of the upstream protein. When beta-lactamase or alkaline phosphatase was linked to the C-terminus of MalE31, the resultant fusion proteins accumulated in an insoluble form, but retained their catalytic activity. In addition, these protein aggregates induced an extracytoplasmic stress response, similar to unfused MalE31. However, using a fluorescent substrate, we found that alkaline phosphatase activity was present inside periplasmic aggregates. These results suggest that periplasmic inclusion body formation may result in intermolecular interactions between participating proteins without loss of function of the fused enzymes.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Periplasma/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , beta-Lactamases/metabolismoRESUMO
A simple way for photochemical patterning of biological molecules onto the inner wall of fused-silica capillary is described. The method is based on a modification of the inner capillary surface with photoactive benzophenone (BP) derivative. The UV irradiation at 365 nm of the capillary filled with a sample solution results in cross-linking of the solutes to the BP moiety via a stable covalent bond. As a proof of concept, oligonucleotides and proteins were arrayed inside the capillary using an inverted microscope as an irradiation device. We demonstrated that the capillary arrays produced in this way are functional and could be used in different bioassays including DNA hybridization, protein interaction studies, and immunoassays. Having a sensitivity comparable to the fluorophore-based assays in a planar format, the capillary array possesses several advantages including submicroliter sample volume and a short assay time. The capillary format should therefore be considered as a possible alternative to a planar format in a number of low-density array applications such as mutation detection and diagnostic immunoassays.
