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Heterologous and Cell-Free Protein Expression Systems

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Plant Genomics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 513))

Summary

In recognition of the fact that a relatively small percentage of ‘named’ genes in databases have any experimental proof for their annotation, attention is shifting towards the more accurate assignment of functions to individual genes in a genome. The central objective will be to reduce our reliance on nucleotide or amino acid sequence similarities as a means to define the functions of genes and to annotate genome sequences. There are many unsolved technical difficulties associated with the purification of specific proteins from extracts of biological material, especially where the protein is present in low abundance, has multiple isoforms or is found in multiple post-translationally modified forms. The relative ease with which cDNAs can be cloned has led to the development of methods through which cDNAs from essentially any source can be expressed in a limited range of suitable host organisms, so that sufficient levels of the encoded proteins can be generated for functional analysis. Recently, these heterologous expression systems have been supplemented by more robust prokaryotic and eukaryotic cell-free protein synthesis systems. In this chapter, common host systems for heterologous expression are reviewed and the current status of cell-free expression systems will be presented. New approaches to overcoming the special problems encountered during the expression of membrane-associated proteins will also be addressed. Methodological considerations, including the characteristics of codon usage in the expressed DNA, peptide tags that facilitate subsequent purification of the expressed proteins and the role of post-translational modifications, are examined.

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Acknowledgements

The financial support of the Australian Research Council, the Grains Research and Development Corporation and the South Australian State Government is gratefully acknowledged. We thank Bianca Kuchel for her invaluable technical assistance with aspects of the work described here.

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  1. Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA, 5064, Australia

    Geoffrey B. Fincher

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  1. Naser Farrokhi
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  2. Maria Hrmova
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  3. Rachel A. Burton
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  4. Geoffrey B. Fincher
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Correspondence to Geoffrey B. Fincher .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Farrokhi, N., Hrmova, M., Burton, R., Fincher, G. (2009). Heterologous and Cell-Free Protein Expression Systems. In: Gustafson, J., Langridge, P., Somers, D. (eds) Plant Genomics. Methods in Molecular Biology™, vol 513. Humana Press. https://doi.org/10.1007/978-1-59745-427-8_10

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    • DOI: https://doi.org/10.1007/978-1-59745-427-8_10

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    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-997-0

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