Mobile gene cassettes collectively carry a highly diverse pool of novel genes, ostensibly for purposes of microbial adaptation. At the sequence level, putative functions can only be assigned to a minority of carried ORFs due to their inherent novelty. Having established these mobilized genes code for folded and functional proteins, the authors have recently adopted the procedures of structural genomics to efficiently sample their structures, thereby scoping their functional range. This chapter outlines protocols used to produce cassette-associated genes as recombinant proteins in Escherichia coli and crystallization procedures based on the dual screen/pH optimization approach of the SECSG (SouthEast Collaboratory for Structural Genomics). Crystal structures solved to date have defined unique members of enzyme fold classes associated with transport and nucleotide metabolism.
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Acknowledgments
The authors are grateful to Moreland Gibbs, Robert Willows, Anwar Sunna, and Meghna Sobti for useful discussions and provision of materials. This work was funded by the Australian Research Council (Discovery scheme), the National Health and Medical Research Council, and Macquarie University. AR acknowledges receipt of a RAACE (Macquarie University) postgraduate scholarship.
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Robinson, A. et al. (2008). Structural Genomics of the Bacterial Mobile Metagenome: an Overview. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_39
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