Anti-GP9 (aa 105-134) polyclonal antibody

Background: Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results: The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions: Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

Neutrophils are the most abundant leukocytes in human blood. Beside being essential responders in bacterial and fungal infections, they also contribute to tissue reactions in many autoimmune and inflammatory diseases. Although several immune responses linked to neutrophil functions have been described to be rhythmic, the mechanism of the circadian regulation of these cells is still not understood. Characterization of the time-of-day-specific control of neutrophil responsiveness could help to better understand the pathomechanism of these inflammatory responses and design effective chronotherapy. Here we report that the time-dependent expression of core clock components in human neutrophils characteristically differs from that in mononuclear cells. Both the low expression and the reduced nuclear accumulation of the essential clock protein BMAL1 suggest that the molecular oscillator is down regulated in neutrophils. By following the expression of the maturation marker Cxcr4 and morphological attributes (side-scattering properties and nuclear segmentation), we found that the distribution of young and aged cells within the peripheral neutrophil pool displays a daily rhythm. In addition, we detected synchronous fluctuations in the plasma level of the CXCR4 ligand CXCL12, an important regulator of cell trafficking within the bone marrow. We found that expression of another maturation marker, the core component of the superoxide generating NADPH oxidase, and parallelly, the superoxide producing capacity of neutrophils were also dependent on the time of the day. In line with this, number of opsonized bacteria engulfed by neutrophils also showed time-dependent differences, supporting that clearance of pathogens shows a daily rhythm. We suggest that maturation-dependent changes in neutrophil responsiveness rather than the cellular autonomous clock are involved in the daily regulation of human neutrophil functions. (C) 2016 Elsevier Inc. All rights reserved.