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Permeabilised Mammalian Cell Systems for Protein Synthesis

  1. Timothy J Tavender

Published Online: 19 MAY 2010

DOI: 10.1002/9780470015902.a0002692.pub2

eLS

eLS

How to Cite

Tavender, T. J. 2010. Permeabilised Mammalian Cell Systems for Protein Synthesis. eLS. .

Author Information

  1. University of Glasgow, Glasgow, UK

Publication History

  1. Published Online: 19 MAY 2010

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 ER-resident 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