Abstract
This chapter describes a method for efficient high-throughput purification of hexahistidine-tagged proteins that are expressed in Escherichia coli (E. coli) using immobilized metal affinity chromatography (IMAC) (2) in a 96-well format. This approach is particularly suitable for proteomic applications that require modest amounts of highly purified proteins to be generated very efficiently. This approach is also very useful for identifying protein targets that are most amenable to scaled-up production for use in structural studies. The typical yield of proteins purified using this system is 50–150 µg, which is generally greater than that of many in vitro expression systems and much less costly. The method as described has been optimized for purifying approx 150 µg of hexahistidine-tagged protein, but the method is flexible, so that the amount of affinity matrix and culture volumes can be adjusted for optimal binding capacity and consequently highest purity. Although the method detailed here uses IMAC to purify hexahistidine-tagged proteins, this basic platform can be used with many other tags and affinity resins.
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Acknowledgments
We thank Shirin Fuller for technical assistance, and Paul Richardson and Peter Beernink for helpful suggestions and critical reading of the manuscript. This work was performed under the auspices of the US Department of Energy, Office of Biological and Environmental Research, by the University of California, Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48, Lawrence Berkeley National Laboratory under contract No. DE-AC03-76SF00098, and Los Alamos National Laboratory under contract No. W-7405-ENG-36.
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© 2005 Humana Press Inc.
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Murphy, M.B., Doyle, S.A. (2005). High-Throughput Purification of Hexahistidine-Tagged Proteins Expressed in E. coli . In: Zanders, E.D. (eds) Chemical Genomics. Methods in Molecular Biology™, vol 310. Humana Press. https://doi.org/10.1007/978-1-59259-948-6_9
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DOI: https://doi.org/10.1007/978-1-59259-948-6_9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-399-2
Online ISBN: 978-1-59259-948-6
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