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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

  • As the market for recombinant proteins continues to skyrocket, biotechs are exploiting a range of options to deal with multiple possibilities. At last month’s CHI “Peptalk” conference, participants explored advances in a number of different expression systems, including bacteria, yeast, mammalian cells, and even cell-free systems.

    A widely used protein-expression system is Pichia pastoris. A number of companies that provide molecular reagents also make plasmids and vectors for manipulating Pichia.

    The focus of VTU Technology, however, is contract development using the Pichia platform, according to Thomas Purkarthofer, Ph.D., head of the company’s R&D biotechnology division. “Our capabilities are based on molecular technologies available in the public domain, combined with a proprietary set of synthetic variants of the Pichia pastoris AOX1 promoter.”

    The AOX1 promoter, inducible with methanol, is known for its power. In order to simplify patent concerns, VTU uses its own optimized plasmids not available publicly, but with no particular IP except for the promoter variants.

    With this approach, the company has developed customized high-performance strains for the production of secreted proteins for use in the pharma, chemical, diagnostics, and agricultural industries. The VTU promoter library of synthetic variants of AOX1 spans a range of activities and expression characteristics in order to match promoter properties and specific expression requirements to a given target protein, as well as efficient expression strains for coexpression of auxiliary proteins required by a particular protocol.

    These strains were isolated and modified individually in order to provide the highest possible level of function. “Our key to success is a combinatorial strategy, matching promoter variants with target and auxiliary genes to maximize protein output,” Dr. Perkarthofer explained.

    The company is in a rather unique situation, in that the technology division was recently formed as a unit of the main company, VTU Holding, which includes engineering and energy-production divisions. This means that there are ample opportunities for collaborative efforts in developing upstream and downstream processes with respect to questions of energy consumption, reactor design, and efficient management of a scale-up program.

    Nothing succeeds like overexpression, as the mammalian-cell protein-expression platforms has so dramatically proven. As scientists, program managers, and regulators have become increasingly more comfortable with mammalian-cell expression programs over the years, it has become virtually impossible to dislodge them from their pinnacle. Nonetheless, alternatives keep appearing, and many have received widespread endorsement.

  • Recombinant Protein Production

    Click Image To Enlarge +
    According to Delphi Genetics, its technology has a number of appealing properties, but perhaps important is the scalibility of the system.

    An interesting bacterial alternative is under development at NIZO Food Research, according to Igor Mierau, Ph.D., project manager for gene expression and fermentation. The company is investigating Lactococcus lactis, a traditional cheese and butter-producing microorganism.

    The bacteria has a number of advantageous properties that lends it to protein-expression platforms. These include absence of endotoxin, inclusion bodies, and sporulation, as well as a range of gene-expression alternatives, within and outside the cell membrane. Moreover, there are a variety of options for control of gene expression, such as nisin, Zn++, and lactic acid-based regulation. To complement the regulatory options, numerous promoters have been described in peer-reviewed literature.

    Dr. Mierau and his colleagues have paid special attention to the nisin system. This 34-amino acid peptide is a bacteriocin, a proteinaceous antibiotic that inhibits the growth of similar or closely related bacterial strains. The nisin-controlled gene expression system (the NICE system) is based on the autoregulation of nisin biosynthesis and drives regulated overproduction of a range of proteins by L. lactis and its cousins.

    According to Dr. Mierau, there is a wealth of literature describing control of gene expression through the NICE system, including overexpression of folate production, expression of a variety of pro- and eukaryotic membrane proteins, bacterial surface antigens, and bioactive peptides.

    Using the plasmid pNZ8150, a wide range of proteins have been expressed in L. lactis, e.g., the antibacterial protein Lysostaphin, with yields in the 300 mg/L range. An easy purification scheme produced 90% pure material.

    Finally, the Mierau group, in collaboration with Edmund Kunji of the Medical Research Council in Cambridge, U.K., has generated eukaryotic membrane proteins, including the yeast mitochondrial carrier protein and the KDEL-receptor 7-helix protein. So while the system may lack some of the panache ascribed to mammalian protein-expression systems, it appears to have promise for specialized projects for which E. coli and other expression systems may not be suitable.

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