Using T7 phage display to select GFP-based binders.

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting. These results demonstrate that specific GFP-based affinity reagents, selected from T7-based libraries, can be used in applications in which only the intrinsic fluorescence is used for detection.

Blocking the NGF-TrkA interaction rescues the developmental loss of LTP in the rat visual cortex: role of the cholinergic system.

Although nerve growth factor (NGF) is a crucial factor in the activity-dependent development and plasticity of visual cortex, its role in synaptic efficacy changes is largely undefined. We demonstrate that the maintenance phase of long-term potentiation (LTP) is blocked by local application of exogenous NGF in rat visual cortex at an early stage of postnatal development. Long-term depression (LTD) and bidirectional plasticity are unaffected. At later postnatal ages, blockade of either endogenous NGF by immunoadhesin (TrkA-IgG) or TrkA receptors by monoclonal antibody rescues LTP. Muscarinic receptor activation/inhibition suggests that LTP dependence on NGF is mediated by the cholinergic system. These results indicate that NGF regulates synaptic strength in well-characterized cortical circuitries.