Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 62, Issue 1, Pages 128-137Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2008.06.014
Keywords
Cytochrome c; High-throughput; Ligation-independent cloning; Periplasmic expression; Protein expression; Shewanella oneidensis
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Funding
- U.S. Department of Energy's Office of Science, Biological and Environmental Research GTL program [DE-AC02-06CH11357]
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Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain-are essential in most organisms. However, they are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. We have addressed this challenge by designing two families of vectors (total of 6 vectors) suitable for ligation-independent cloning and developing a pipeline for expression and solubility analysis of cytochromes c. This stem has been validated by expression analysis of thirty genes from Shewanella oneidensis coding for cytochromes c or cytochromes c-type domains predicted to have 1-4 hemes. Out of 30 targets, 26 (87%) were obtained in soluble form in one or more vectors. This work establishes a methodology for high-throughput expression of this class of proteins and provides a clone resource for the microbiological and functional genomics research communities. (C) 2008 Elsevier Inc. All rights reserved.
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