Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmids pET-21d (+). Hexa-histidine tagged apo-alpha and apo-beta subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of Escherichia coli cells with phycoerythrobilin. Subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. Addition of phycoerythrobilin to cells did not show cytotoxic effects in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, Rphycoerythrin apo-subunits were fused to maltose binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly-fluorescent soluble fusion proteins were formed containing phycoerythrobilin as the sole chromophore. Fusion proteins were localized by fluorescence microscopy either throughout Escherichia coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to ten times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

No abstract

Periods of intense physical training are associated with an increased incidence of infection. We hypothesize that the TLR pathways involved in the initial response to multiple pathogens are compromised during intense physical exercise stress. In order to test this hypothesis, eight male collegiate wrestlers participated in one month of intense physical conditioning stress. Blood was collected at rest both before and after a 1 month period of intense exercise, and after completing a single 90 minute session of exhaustive exercise. TLR receptor pathway expression was assessed by microarray. The results showed that 1 month of intense exercise conditioning decreased expression of genes that may be important in antiviral defense (IFN-alpha, TLR3, PKRKA, SARM1), and inflammatory response (TNFSRF, PTGS2, REL). Ninety minutes of intense exercise decreased IFN-gamma and IRF1, but expression of TLR pathways associated with anti-bacterial defense increased (TLR5, TIRAP, PGLYRP2, LY86). These findings are consistent with epidemiological data suggesting an increased incidence of respiratory viral infections during periods of intense physical exercise stress. The results also implicate several genes important in inflammation, and further research is needed to determine whether these genes play a significant role in the recently reported anti-inflammatory effects of repeated exercise.