Page 34 - Macromolecular Crystallography

CHAPTER 2

High-throughput cloning, expression,

and purification

Raymond J. Owens, Joanne E. Nettleship, Nick S. Berrow,
Sarah Sainsbury, A. Radu Aricescu, David I. Stuart, and
David K. Stammers

2.1 Introduction be carried out manually with appropriate equip-
ment, for example multichannel pipette dispensers.
High-throughput sequencing of eukaryotic, viral,
The motivation to implement automation is largely
and bacterial genomes is providing a huge database
to enable processes to be scaleable and sustainable
of proteins with potential for structure–function
as error-free operations. In this article we review the
analysis. In response to this opportunity, structural
technical developments that have come from struc-
genomics projects have been initiated world-wide
tural proteomics and provide protocols for carrying
with the aim of establishing high-throughput struc-
out cloning, expression, and purification procedures
ture determination on a genome-wide scale. Crucial
in a relatively high-throughput (HTP) and parallel
to this effort has been the development of protein
approach.
production technologies for the high-throughput
cloning, expression, and purification of proteins.
Large-scale structural genomic projects were initi- 2.2 Cloning
ated in the US by the National Institute of Health
(NIH) and in Japan by the Riken Laboratory from Two options are available for constructing the
1998 to 2000. European projects followed, includ- expression vectors required for protein produc-
ing the Protein Structure Factory in Berlin (www. tion, namely ligation-dependent and ligation-
proteinstrukturfabrik.de), Oxford Protein Produc- independent cloning (LIC). The former makes use
tion Facility (OPPF) (www.oppf.ox.ac.uk), and the of standard restriction enzyme digestion in combi-
EU-sponsored Structural Proteomics In Europe nation with DNA ligation to produce the vectors.
(SPINE: www.spineurope.org) programme. The Whereas the latter utilizes either some form of
scale of these projects has been smaller than the recombination event or the production and anneal-
US/Japan initiatives, with a focus at the outset on ing of single-stranded overhangs, both of which
human and viral targets. For all projects, there has avoid the need to restriction digest the input DNA.
been an emphasis on parallel processing, both in Typically, in both cases the starting DNA is a PCR
termsofmolecularcloning, expression, andpurifica- product corresponding to the whole or part of an
tion, driven by the need to accommodate relatively open reading frame (ORF) produced from either
large numbers of potential targets for structural a genomic or cDNA template. The PCR primers
biology at an acceptable cost. This has led to vary- incorporate either restriction enzyme recognition
ing degrees of automation and most of the groups sites or the sequences required for LIC reactivity.
involved have set up semiautomated liquid han- By using rare cutting restriction enzyme sites, lig-
dling systems to carry out some or all of their ation based cloning has been used effectively for
protocols. However, the protocols can equally well semi-automated high-throughput cloning (Lesley

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