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      A Novel Disulfide-Rich Protein Motif from Avian Eggshell Membranes

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          Abstract

          Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X 4- C-X 5- C-X 8- C-X 6 pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a- b modules; or an a- b- b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch ( Taeniopygia guttata) and in the oviparous green anole lizard ( Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a- b- a- b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

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          Large numbers of MS/MS peptide spectra generated in proteomics experiments require efficient, sensitive and specific algorithms for peptide identification. In the Open Mass Spectrometry Search Algorithm (OMSSA), specificity is calculated by a classic probability score using an explicit model for matching experimental spectra to sequences. At default thresholds, OMSSA matches more spectra from a standard protein cocktail than a comparable algorithm. OMSSA is designed to be faster than published algorithms in searching large MS/MS datasets.
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              A method for efficient isotopic labeling of recombinant proteins.

              A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                30 March 2011
                : 6
                : 3
                : e18187
                Affiliations
                [1 ]Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States of America
                [2 ]Department of Chemistry, Towson University, Towson, Maryland, United States of America
                University of Crete, Greece
                Author notes

                Conceived and designed the experiments: VKK SAG SR TP CT. Performed the experiments: VKK SAG SP SR. Analyzed the data: VKK SAG SP TP CT. Wrote the paper: VKK SAG TP CT.

                Article
                PONE-D-11-00760
                10.1371/journal.pone.0018187
                3068167
                21479176
                1da9dcfd-0756-4f91-a877-e27f5b7ec9cb
                Kodali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 1 January 2011
                : 22 February 2011
                Page count
                Pages: 11
                Categories
                Research Article
                Biology
                Biochemistry
                Proteins
                Extracellular Matrix Proteins
                Protein Chemistry
                Protein Classes
                Protein Structure
                Recombinant Proteins
                Structural Proteins
                Macromolecular Assemblies
                Biophysics
                Protein Folding

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                Uncategorized

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