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ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN ESCHERICHIA COLI WHEN EXPOSED TO L‐TELLUROMETHIONINE
Author(s) -
Broderick Kathleen Marie,
Boles Jeffrey O,
Brinkley Sarah
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.776.3
Subject(s) - proteome , escherichia coli , amino acid , methionine , peptide , two dimensional gel electrophoresis , peptide mass fingerprinting , gel electrophoresis , biochemistry , chemistry , biology , auxotrophy , mass spectrometry , proteomics , chromatography , microbiology and biotechnology , gene
Analyzing the three‐dimensional structure of a protein using x‐ray crystallography can be improved by incorporating unnatural amino acids into that protein. Most naturally occurring atoms do not have sufficient mass to diffract electrons. Using unnatural amino acids provides a solution to this problem. Bioincorporation of L‐Telluromethionine can provide residues with sufficient mass for electron scattering using a very simple procedure. However telluromethionine has cytotoxic effects which reduce cell growth and uptake. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. coli DL41(DE3) (pCock) expression system. Both telluromethionyl and methionyl exposed cultures were analyzed with the objective of identifying differentially expressed proteins. Analysis was carried out using 2D gel electrophoresis followed by tryptic digestion, and peptide fragment analysis using nano‐ESI/qTOF/MS. Cell cultures harvested at the beginning of the stationary phase of growth are compared to cells harvested at the maximal expression (with and without exposure to TeMet). In addition, the proteome is also examined using ion exchange chromatography to fractionate the proteome prior to 2D gel analysis. The biological roles of differentially expressed proteins will be elucidated and applied to improving methods for the bioincorporation of telluromethionine.