Therapeutic Proteins

Volume 308 of the series Methods in Molecular Biology™ pp 99-106

Site-Specific Integration for High-Level Protein Production in Mammalian Cells

  • Bhaskar ThyagarajanAffiliated withPoetic Genetics, LLC
  • , Michele P. CalosAffiliated withDepartment of Genetics, Stanford University School of Medicine

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Recombinant proteins have various applications in basic and applied research, as well as in therapy. Numerous systems have been developed to produce large protein amounts (13). The most convenient systems are those that involve the overexpression of recombinant proteins in bacteria, mainly because they are fast and easy. However, there is a need for eukaryotic overexpression systems, because several proteins require either the proper folding environment or posttranslational modifications for physiological activity (4). The ideal system for eukaryotic overexpression would have integration of the expression cassette in the genome of the target cell at a location that permits strong and long-term expression. However, most current methods for achieving integration of plasmid DNA into the genome involve random integration of DNA (58). Expression of the recombinant protein in clones generated by such methods is dependent on the chromatin context at the site of integration and often leads to low expression levels (9,10). Therefore, many clones should be screened to find those expressing the protein of interest at the desired level.