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Feb 1, 2009 (Vol. 29, No. 3)

Alternatives to Mammalian Expression

Bacterial, Yeast, and Cell-Free Systems Are among Options Attempting to Replace Perennial Favorite

  • Isotopic Enrichment

    “Bacteria can be coaxed into producing just one protein,” says Masayori Inouye, Ph.D., professor at the Robert Wood Johnson Medical School. For a number of years, Dr. Inouye and his colleagues have been studying a bacterial toxin that can be used to turn E. coli into a factory producing a single protein of interest.

    The Inouye group has evaluated MazF, an endoribonuclease that degrades only messenger RNA by cleaving it at a specific nucleotide site. The researchers designed an E. coli strain that carried this gene, they then followed up by adding a protein-producing gene of interest, which lacked the specific cleavage site. When the MazF gene is turned on, all other protein synthesis grinds to a halt, since all the other messengers are rapidly degraded. The overwhelming amount of protein produced is now the target protein, which can be specifically labeled by adding 13C and 15N to the culture medium.

    Dr. Inouye envisions the technique as a means of producing pure protein easily and rapidly for structural and functional studies of proteins in intact, living cells using nuclear magnetic resonance.

    The Inouye platform, referred to as SPP (single protein production), can be adapted to maximize production of virtually any protein, either membrane bound or cytoplasmic, according to the group. This approach to dealing with protein purification allows the investigator to forego the use of affinity columns or other costly materials and hardware.  Moreover, it allows for structural studies to be conducted on proteins that are toxic to other systems.

    The genes that constitute the platform can be transferred to eukaryotic systems, including yeast and mammalian cells. Given the need for industrial-scale protein-purification technology that can escape the burden of complex resins, columns, and ligands, the technology has great intrinsic appeal.

    In the bioprocessing field, all the low-hanging fruit was carted away long ago, so it is a tribute to the biotechnologists profiled in these pages that they were able to address longstanding problems in a fresh and creative fashion. If these technologies prove to be acceptable on a large scale, they could make major inroads into areas of production and purification that have so far resisted easy solutions, and have relied upon time-tested but laborious and expensive technologies. 


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