Premium
Improving telluromethionyl uptake, microbial growth and recombinant protein expression
Author(s) -
Boles Jeffrey Oakley,
Brinkley Sarah,
Mancuso Matthew
Publication year - 2011
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.25.1_supplement.926.1
Subject(s) - recombinant dna , auxotrophy , chemistry , proteome , escherichia coli , biochemistry , protein expression , amino acid , protein biosynthesis , methionine , gene expression , target protein , gene , biology
Biosynthetic incorporation of tellurium into proteins in the form of telluromethionine has shown to be advantageous for determining protein structure, however, cell growth and recombinant protein expression are often not reproducible. The three‐dimensional conformation of proteins can be solved with greater ease by utilizing unnatural amino acids (UAA) since the phasing atom is an in situ isomorphous replacement. Unfortunately, cytotoxicity of TeMet substantially reduces in vivo uptake as E. coli is very sensitive to tellurite (a decomposition by‐product). Differential expression of the Met auxotroph, Eschericia coli DL41, revealed differences between cells forced to use TeMet in the translation of proteins and cells grown in an authentic methionine environment. Proteome detection with UPLC/qTOF MS enabled the identification of proteins differentially expressed providing insight into the consequences of growth and expression in the presence of TeMet. A modified growth medium centered around the minimization of differential expression, coupled with the utilization of a tellurite resistance gene, provides a potential new tool for the elucidation of 3D structure. Support is acknowledged from the National Science Foundation.