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Macromolecular Crystallographyconventional and high-throughput methods$
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Mark R. Sanderson and Jane V. Skelly

Print publication date: 2007

Print ISBN-13: 9780198520979

Published to Oxford Scholarship Online: September 2007

DOI: 10.1093/acprof:oso/9780198520979.001.0001

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High-throughput cloning, expression, and purification

High-throughput cloning, expression, and purification

(p.23) CHAPTER 2 High-throughput cloning, expression, and purification
Macromolecular Crystallography

Raymond J. Owens

Joanne E. Nettleship

Nick S. Berrow

Sarah Sainsbury

A. Radu Aricescu

David I. Stuart

David K. Stammers

Oxford University Press

High-throughput sequencing of eukaryotic, viral, and bacterial genomes provides a huge database of proteins with potential for structure-function analysis. In response to this opportunity, structural genomics projects have been initiated world-wide with the aim of establishing high-throughput structure determination on a genome-wide scale. Crucial to this effort has been the development of protein production technologies for the high-throughput cloning, expression, and purification of proteins. Large-scale structural genomic projects were initiated in the US and Europe, and all have emphasized parallel processing, both in terms of molecular cloning, expression, and purification, driven by the need to accommodate relatively large numbers of potential targets for structural biology at an acceptable cost. This has led to varying degrees of automation and most of the groups involved have set up semiautomated liquid handling systems to carry out some or all of their protocols. However, the protocols can equally well be carried out manually with appropriate equipment, for example multichannel pipette dispensers. The motivation to implement automation is largely to enable processes to be scaleable and sustainable as error-free operations. This chapter reviews the technical developments that have come from structural proteomics and provides protocols for carrying out cloning, expression, and purification procedures in a relatively high-throughput (HTP) and parallel approach.

Keywords:   structural proteomics, cloning, expression, purification, genome sequencing

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