Overview of Protein Expression by Mammalian Cells

David Gray1

1 Chiron Corporation, Emeryville, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.9
DOI:  10.1002/0471140864.ps0509s10
Online Posting Date:  May, 2001
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Abstract

This unit reviews the stages involved in protein production in mammalian cells using a stable‐expression approach. Choice of cell type is discussed, as is transfection of the host cells, methods for selection and amplification of transformants, and growth of cells at appropriate scale for protein production. Since post‐transcriptional modification and intracellular protein transportation are important features of recombinant‐protein production in mammalian cells, some description of these mechanisms is included.

     
 
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Table of Contents

  • Choice of Mammalian Cell Host
  • Transfection, Selection, and Amplification
  • Protein Translation, Quality Control, and Covalent Modification
  • Growth of Mammalian Cells for Protein Expression
  • Scale of Operation
  • Summary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •   FigureFigure 5.9.1 Flow chart of activities in the development of a mammalian‐cell expression system.
  •   FigureFigure 5.9.2 Representation of a mammalian expression vector based on the vector pRSC, published by Tsang et al. (); adapted with permission. The plasmid has two multiple cloning sites (MCSs) and three promoters. One promoter drives the neomycin‐resistance protein while the remaining two MCSs have specific promoters and can be used to incorporate an amplification marker (DHFR) and a gene for the protein of interest.
  •   FigureFigure 5.9.3 Intracellular protein translation and cisternal transport. Cytoplasmic translation results in a nascent polypeptide that is chaperoned by the hsp70 protein, which may direct the ribosome‐peptide complex to the mitochondrion or the ER upon N‐terminal peptide signaling. A signal‐recognition particle (SRP) binds to the signal sequence, halting translation until it has docked at the SRP receptor. The hsp70 protein maintains the protein in a translocation‐competent state, enabling membrane protrusion. Inside the ER the protein undergoes refolding assisted by protein disulfide isomerase (PDI), BiP, and calnexin (CXN). Initial N‐glycosylation takes place cotranslationally in the ER. Misfolded protein, associated with BiP, is retained and eventually degraded. Transport of folded, assembled protein between the ER and Golgi cisternae occurs through a series consisting of budding, vesicle formation, and specific binding to target cisternae surfaces, through to the trans Golgi (TGN), where distribution to particular destinations occurs. Abbreviations: ARF, ADP‐ribosylation factor; COPs, a family of coat proteins; Dol, dolichol; endo H, endoglycosidase H; GTP bp, GTP binding protein; NSF, NEM‐sensitive cytosolic factor (identical to yeast sec 18p); SNAPs, three soluble NSF attachment proteins; v‐SNARE and t‐SNARE, SNAP receptors. Symbols: solid circles, N‐acetyl glucosamine; open circles, mannose; solid diamonds, glucose; open squares, galactose; solid squares, sialic acid; solid triangles, fucose; circled 1, α‐glucosidase I; circled 2, α‐glucosidase II; circled 3, α‐1,2‐mannosidase; circled 4, GlcNAc transferase I; circled 5, α‐mannosidase II; circled 6, GlcNAc transferase II; circled 7, α‐1,6‐fucosyl transferase; circled 8, β‐1,4‐galactose transferase; and circled 9, α‐2,3‐sialyl transferase.

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Literature Cited

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