Addressing Shewanella oneidensis "cytochromome": the first step towards high-throughput expression of cytochromes c.
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 system 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.
Londer
Yuri Y
YY
Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA. londer@neb.com
Giuliani
Sarah E
SE
Peppler
Terese
T
Collart
Frank R
FR
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D019897:Periplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020592:Shewanella / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D045304:Cytochromes c