Eukaryotic expression: developments for structural proteomics.
Journal article, 2006
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.
Baculoviridae
Cultured
metabolism
Selenomethionine
Cloning
diagnostic use
Proteomics
Yeasts
metabolism
Eukaryotic Cells
Gene Expression
Animals
genetics
Cells
Molecular
methods
Glycosylation
Author
A R Aricescu
Wellcome Trust Centre for Human Genetics
R Assenberg
Wellcome Trust Centre for Human Genetics
Joakim Norbeck
Aston University
Chalmers
D Busso
IGBMC Institut de Genetique et de Biologie Moleculaire et Cellulaire
V T Chang
John Radcliffe Hospital
S J Davis
John Radcliffe Hospital
A Dubrovsky
Karolinska Institutet
Lena Gustafsson
Chalmers, Chemical and Biological Engineering
Kristina Hedfalk
Chalmers, Chemical and Biological Engineering, Molecular Biotechnology
U Heinemann
The Max Delbrück Center for Molecular Medicine
I M Jones
University of Reading
D Ksiazek
Max Planck Society
C Lang
Technische Universität Berlin
K Maskos
Max Planck Society
A Messerschmidt
Max Planck Society
S Macieira
Max Planck Society
Y Peleg
Weizmann Institute of Science
A Perrakis
The Netherlands Cancer Institute
A Poterszman
IGBMC Institut de Genetique et de Biologie Moleculaire et Cellulaire
G Schneider
John Radcliffe Hospital
T K Sixma
The Netherlands Cancer Institute
J L Sussman
Weizmann Institute of Science
G Sutton
Wellcome Trust Centre for Human Genetics
N Tarboureich
Institut Laue-Langevin
T Zeev-Ben-Mordehai
Weizmann Institute of Science
E Yvonne Jones
Wellcome Trust Centre for Human Genetics
Acta Crystallographica Section D: Biological Crystallography
0907-4449 (ISSN) 1399-0047 (eISSN)
Vol. 62 Pt 10 1114-24Subject Categories
Biological Sciences
DOI
10.1107/S0907444906029805
PubMed
17001089