Permeabilised Mammalian Cell Systems for Protein Synthesis

Abstract

The use of semipermeabilised cells allows the study of complex intracellular processes such as protein translocation, folding, glycosylation, disulfide bond formation and degradation in a system amenable to experimental manipulation. Semipermeabilised cells can be combined with an in vitro translation system to synthesise endoplasmic reticulum (ER) targeted proteins from custom made messenger ribonucleic acid (mRNA). The ability to manipulate the translated protein means that the effect of changes to the primary amino acid sequence on protein folding and assembly can be readily assessed. Semipermeabilised cells can be prepared from cell lines expressing elevated or diminished levels of ERÔÇÉresident proteins and can also be pretreated with chemical modifiers of ER processes. Using such approaches, it is possible to examine the influence of various ER pathways on folding and posttranslational modification of individual secretory proteins.

Key concepts:

  • Semipermeabilised cells possess an intact, functional endoplasmic reticulum and are free of cytosol and endogenous mRNA.

  • Secretory proteins translated in the presence of semipermeabilised cells are translocated and posttranslationally modified as in intact cells.

  • Use of an in vitro translation system allows complete user manipulation of translated protein sequences.

Keywords: translation; protein synthesis; posttranslational modification; translocation; endoplasmic reticulum

Figure 1.

Schematic illustrating key stages during preparation of semipermeabilised mammalian cells. Intact cells in suspension (a) are treated with digitonin leading to permeabilisation of the plasma membrane (b). Subsequent wash steps and nuclease treatment remove endogenous cystosolic components and mRNA (c). Resultant semipermeabilised cells can be supplemented into an in vitro translation or coupled transcription/translation system (d). In this case, reticulocyte lysate acts as an artificial cytosol, whereas synthesised secretory proteins can be targeted to, and translocated into, the intact endoplasmic reticulum.

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References

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Further Reading

Bulleid NJ, Dalley JA and Lees JF (1997) The C‐propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis. EMBO Journal 16: 6694–6701.

Farmery MR, Allen S, Allen AJ and Bulleid NJ (2000) The role of ERp57 in disulfide bond formation during the assembly of major histocompatibility complex class I in a synchronized semipermeabilized cell translation system. Journal of Biological Chemistry 275: 14933–14938.

Jessop CE, Chakravarthi S, Garbi N et al. (2007) ERp57 is essential for efficient folding of glycoproteins sharing common structural domains. EMBO Journal 26: 28–40.

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How to Cite close
Tavender, Timothy J(May 2010) Permeabilised Mammalian Cell Systems for Protein Synthesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002692.pub2]