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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

  • Elastin-Like Biopolymers

    There is an insatiable demand for new protein-purification platforms. A notable contribution to this gaggle of approaches is based on the use of elastin-like biopolymers, as described by Ariel Boutaud, Ph.D., director of research at Phase Bioscience.

    Much of the protein-purification technology within the biotech industry is based on the use of affinity chromatography, in which an expressed recombinant protein is fused to a peptide having high affinity for a particular ligand. A resin column is constructed with the ligand coupled to it, and the crude protein soup is passed through the column, leaving the target protein bound to the column. Subsequently the peptide tail can be removed, leaving the protein target.

    While this approach is ubiquitous throughout the industry, it has notable shortcomings, including high cost and the need for specialized equipment and expensive resins. Scale-up to industrial levels is a hit-or-miss proposition, frequently frustrating and expensive, and yields can be paltry with significant loss.

    The deltaPhase recombinant expression/purification system is based on the transition properties of elastin-like polymers and their ability to retain this inverse temperature-phase transition when conjugated to other molecules. The phase transition offers a new method of purification for therapeutic proteins and peptides. The process consists of fusing an ELP sequence to the N or C terminus of the polypeptide of interest by recombinant DNA techniques. The DNA coding for the polypeptide or protein tagged with an ELP is introduced into an expression system (e.g., E.  coli or mammalian cells) for production.

    Once the protein ELP fusion is produced, the cells are lysed in the case of an E. coli expression system or the culture supernatant is collected for a mammalian-expression platform. The ELP fusion protein is then phase transitioned to form insoluble aggregates, which are isolated by centrifugation or filtration. After isolating the aggregated ELP protein, it is resolubilized by decreasing the temperature. The ELP may then be cleaved enzymatically from the fusion protein. Another cycle of phase transition purification will separate any uncleaved product from the ELP, which remains in the insoluble fraction and leaves behind the purified product in the soluble fraction.

    “We believe that the deltaPhase technology offers an innovative method for protein/polypeptide purification and avoids costly chromatography,” Dr. Boutaud stated. “We would argue that PhaseBio’s deltaPhase technology is the first new production and purification method developed in the last 20 years.”

  • Freedom from Antibiotics

    Click Image To Enlarge +
    VTU Technology offers protein-expression services with the methylotrophic yeast Pichia pastoris.

    E. coli is recognized as the workhorse of recombinant DNA manipulation. A long-standing feature of this technology is the requirement for antibiotics during the clonal selection and protein-expression phases, in order to stabilize the plasmids that carry the essential genetic information. This need for antibiotics represents a substantial impediment to the straightforward manipulation of bacterial strains and vectors, according to Philippe Gabant, Ph.D., CEO and founder at Delphi Genetics. Moreover, regulatory questions make the use of antibiotic-resistance factors undesirable and under the best of circumstances, plasmid instability can frequently lead to low yields and introduce unwanted variability during manufacturing.

    To confront this problem the company took advantage of its expertise in bacterial poison antidote genes. This is a recently developed selection and stabilization tool, based on naturally occurring poison-antidote systems. As Dr. Gabant explains, the selection gene targets DNA replication, an essential bacterial process, specifically the enzyme DNA gyrase. If the selection gene is unleashed, it shuts off DNA gyrase and the cell is doomed. The antidote protein interacts with the selection protein and blocks its lethal activity.

    “This constitutes a powerful alternative to antibiotics for clone selection and plasmid stabilization,” Dr. Gabant stated.

    In designing the production strains, the selection gene is integrated into the bacterial chromosome, but it fails to kill the cell as long as it is repressed by the antidote gene, which is engineered into the plasmid. So without the use of antibiotics the strain is stabilized for a variety of different applications. The plasmid, pStaby1.2, is available with or without antibiotic-resistance genes. It performs admirably with protein-production levels and plasmid DNA levels 3 to 5 times higher than in the case of strains lacking the bacterial selection system, Dr. Gabant added.

    The Delphi technology has a number of appealing properties, but perhaps the most important is the scalability of the system. Any E. coli-based protein-production system can be upgraded to industrial levels in which the necessity of antibiotics to keep the genes in place is no longer a consideration. Not only does this constitute a major technical simplification, but the cost savings involved in the omission of antibiotics from thousands of liters of medium are substantial.


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