Eukaryotic expression: developments for structural proteomics

Aricescu AR, Assenberg R, Bill RM, Busso D, Chang VT, Davis SJ, Dubrovsky A, Gustafsson L, Hedfalk K, Heinemann U, Jones IM, Ksiazek D, Lang C, Maskos K, Messerschmidt A, Macieira S, Peleg Y, Perrakis A, Poterszman A, Schneider G, Sixma TK, Sussman JL, Sutton G, Tarboureich N, Zeev-Ben-Mordehai T, Jones EY. (2006), Acta Crystallogr D Biol Crystallogr. 62, 1114-24

Abstract

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.

Key figure: Deglycosylation of the receptor tyrosine phosphatase RPTPμ expressed transiently in 293T and 293S/GnT1^−/− cells

5 mg of purified protein was treated with 250 U of endoglycosidase (EndoH) at pH 5.2 for 6 h at 310 K in each case. The samples were then analysed by SDS–PAGE under reducing conditions; the band marked with an asterisk is EndoH. Expression of RPTPμ in 293S/GnT1^−/− cells leads to a larger fraction of the protein being `nicked’. In contrast to the partial EndoH-sensitivity of the 293T-derived material, the 293S/GnT1^−/−-derived protein is completely EndoH-sensitive.