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The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Original publication

DOI

10.1107/S0907444906029805

Type

Journal article

Journal

Acta Crystallogr D Biol Crystallogr

Publication Date

10/2006

Volume

62

Pages

1114 - 1124

Keywords

Animals, Baculoviridae, Cells, Cultured, Cloning, Molecular, Eukaryotic Cells, Gene Expression, Glycosylation, Proteomics, Selenomethionine, Yeasts