Eukaryotic expression: developments for structural proteomics

A. R. Aricescu, R. Assenberg, R. M. Bill, D. Busso, V. T. Chang, S. J. Davis, A. Dubrovsky, L.L. Gustafsson, K. Hedfalk, U. Heinemann, I. M. Jones, D. Ksiazek, Chim C Lang, K. Maskos, A. Messerschmidt, S. Macieira, Y. Peleg, A. Perrakis, A. Poterszman, G. SchneiderTitia K Sixma, Joel L. Sussman, G. Sutton, N. Tarboureich, T. Zeev-Ben-Mordehai, E. Yvonne Jones

Research output: Contribution to journalArticleAcademicpeer-review

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.
Original languageEnglish
Pages (from-to)1114-1124
Number of pages11
JournalActa Crystallographica Section D: Biological Crystallography
Volume62
DOIs
Publication statusPublished - Oct 2006
Externally publishedYes

Keywords

  • high throughput
  • eukaryotic expression
  • baculovirus
  • yeast
  • mammalian cells

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