High-throughput production of Pyrococcus furiosus proteins: Considerations for metalloproteins
Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes âˆ¼2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by over-expression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors. Â© 2005 International Union of Crystallography Printed in Great Britain - all rights reserved.
Journal of Synchrotron Radiation
Jenney, Francis E. Jr.; Brereton, P. S.; Izumi, M.; Poole, F. L. II; Shah, C.; Sugar, F. J.; Lee, H.; and Adams, M. W., "High-throughput production of Pyrococcus furiosus proteins: Considerations for metalloproteins" (2005). PCOM Scholarly Papers. 590.
This article was published in Journal of Synchrotron Radiation, Volume 12, Issue 1, Pages 8-12.The published version is available at http://dx.doi.org/10.1107/S0909049504027840.
Copyright © 2005 IUCr.