Open AccessEukaryotic expression: developments for structural proteomics
Author(s) -
Aricescu A. R.,
Assenberg R.,
Bill R. M.,
Busso D.,
Chang V. T.,
Davis S. J.,
Dubrovsky A.,
Gustafsson L.,
Hedfalk K.,
Heinemann U.,
Jones I. M.,
Ksiazek D.,
Lang C.,
Maskos K.,
Messerschmidt A.,
Macieira S.,
Peleg Y.,
Perrakis A.,
Poterszman A.,
Schneider G.,
Sixma T. K.,
Sussman J. L.,
Sutton G.,
Tarboureich N.,
ZeevBenMordehai T.,
Jones E. Yvonne
Publication year - 2006
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444906029805
Subject(s) - proteomics , cloning (programming) , pichia pastoris , saccharomyces cerevisiae , yeast , biology , computational biology , protein expression , glycosylation , microbiology and biotechnology , pichia , protein biosynthesis , gene , biochemistry , recombinant dna , computer science , programming language
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.