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Methionine sulfoxide (MetO) is a common posttranslational modification to proteins occurring in vivo.These modifications are prevalent when reactive oxygen species levels are increased. To enable the detection of MetO in pure and extracted proteins from various sources, we have developed novel antibodies that can recognize MetO-proteins. These antibodies are polyclonal antibodies raised against an oxidized methionine-rich zein protein (MetO-DZS18) that are shown to recognize methionine oxidation in pure proteins and mouse and yeast extracts. Furthermore, mouse serum albumin and immunoglobulin (IgG)were shown to accumulate MetO as function of age especially in serums of methionine sulfoxide reductase A knockout mice. Interestingly, high levels of methionine-oxidized IgG in serums of subjects diagnosed with Alzheimer's disease were detected by western blot analysis using these antibodies. It is suggested that anti-MetO-DZS18 antibodies can be applied in the identification of proteins that undergo methionine oxidation under oxidative stress, aging, or disease state conditions.

In the present work, we developed the Fc-III tagged protein expression system for protein purification and detection. The Fc-III sequence encodes for a 13 residue peptide and this peptide is cyclized by disulfide bond formation when the fusion protein is expressed. The Fc-III-fusion proteins selectively bind to immunoglobulin Fc domains (IgG-Fc) expressed from E. coli. We showed the efficient purification of Fc-III tagged proteins by immobilized non-native IgG- Fc and the detection of the cellular locations of fusion proteins by fluorescent-conjugated IgG-Fc. Our results prove that Fc-III tagged protein expression system is a simple and efficient tool for protein purification and detection and is a useful addition to the biochemistry and proteomics toolbox. [ABSTRACT FROM AUTHOR]

The proteome of the marine bacterium Photobacterium angustum S14 was exposed to UVB and analyzed by the implementation of both the post-digest ICPL labeling method and 2D-DIGE technique using exponentially growing cells. A total of 40 and 23 proteins were quantified in all replicates using either the ICPL or 2D-DIGE methods, respectively. By combining both datasets from 8 biological replicates (4 biological replicates for each proteomics technique), 55 proteins were found to respond significantly to UVB radiation in P. angustum. A total of 8 UVB biomarkers of P. angustum were quantified in all replicates using both methods. Among them, the protein found to present the highest increase in abundance (almost a 3-fold change) was RecA, which is known to play a crucial role in the so-called recombinational repair process. We also observed a high number of antioxidants, transport proteins, metabolism-related proteins, transcription/ translation regulators, chaperonins and proteases. We also discuss and compare the UVB response and global protein expression profiles obtained for two different marine bacteria with trophic lifestyles: the copiotroph P. angustum and oligotroph Sphingopyxis alaskensis. [ABSTRACT FROM AUTHOR]

Many lines of evidence suggest an interaction between glycosaminoglycans (GAGs) and the PrP proteins as well as a possible role for GAGs in prion disease pathogenesis. In this work, we sought to determine whether the PrP. GAG interaction affects the incorporation of PrPSc (the scrapie isoform of PrP) to normal cells. This may be the first step in prion disease pathogenesis. To this effect, we incubated proteinase K-digested hamster scrapie brain homogenates with several lines of Chinese hamster ovary (CHO) cells in the presence or absence of heparin. Our results show that over a large range of PrPSc concentrations the binding of PrPSc to wild type CHO cells, which do not express detectable PrP, was equivalent to the binding of PrPSc to CHO cells overexpressing PrP. A significant part of PrPSc binding to both lines could be inhibited by heparin. Additional evidence that PrPSc binding to cells was dependent on the presence of GAGs could be concluded from the fact that the binding of PrPSc to CHO cells missing GAGs on the cell surface was significantly reduced. Interestingly, preincubation of scrapie brain homogenate with heparin before intraperitoneal inoculation into normal hamsters resulted in a significant delay in prion disease manifestation. [ABSTRACT FROM AUTHOR]