Chapter

Classical cloning, expression, and purification

Jane Skelly, Maninder K. Sohi and Thil Batuwangala

in Macromolecular Crystallography

Published in print August 2007 | ISBN: 9780198520979
Published online September 2007 | e-ISBN: 9780191706295 | DOI: http://dx.doi.org/10.1093/acprof:oso/9780198520979.003.0001
 Classical cloning, expression, and purification

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The ideal protein-expression strategy for X-ray structural analysis should provide correctly folded, soluble, and active protein in sufficient quantities for successful crystallization. Subsequent isolation and purification must be designed to achieve a polished product as rapidly as possible, involving a minimum number of steps. The simplest and least expensive methods employ bacterial hosts such as Escherichia coli, Bacillus, and Staphylococcus but if the target protein is from an eukaryotic source requiring post-translational processing for full functionality, an eukaryotic vector-host system would be appropriate. This chapter discusses the processes of cloning and expression, and protein extraction and isolation.

Keywords: X-ray structural analysis; crystallization; cloning; expression; protein extraction; protein isolation

Chapter.  13467 words. 

Subjects: Biochemistry

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