Sidebar: Not Just for Recombinant Proteins
The more that is learned about cell genetics, the greater the ability to manipulate all sorts of production cells. Researchers from the University at Buffalo have discovered the most efficient production system yet for isoflavonoids, multiring organic molecules of great interest in pharmaceuticals.
Mattheos Koffas, Ph.D., associate professor in the department of chemical and biological engineering, has engineered brewer’s yeast to produce isoflavonoids at titers of 50 mg/L. These quantities at first seem meager in light of PER.C6 productivity of 40 grams per liter, but Dr. Koffas notes that early fermentations for polyketide antibiotic manufacturing yielded only about 1 mg/L of product.
The technique opens up new avenues to the synthesis of thousands of natural and non-natural isoflavonoids, says Dr. Koffas, whose group has optimized the yeast for the combined action of three isoflavonoid-synthesizing enzymes using common flavanone precursors. Several compounds have already shown activity in estrogen receptor binding, an attribute of several marketed breast cancer therapies.
The required enzymes—P450 monooxygenases, p45 reductase, and a dehydrogenase—are normally found in plants, where they naturally produce isoflavonoids in excruciatingly low concentrations. Screened enzymes were inserted into the yeast, and the organisms were selected for optimal productivity. The organisms are versatile, producing a wide variety of products depending on the starting flavanone supplied.
Creating cell- (vs. egg-) derived influenza vaccines has focused on several cell types that grow flu virus effectively. Viruses are particularly fond of one such line, Madin Darby canine kidney (MDCK) cells, but MDCK cells are stubbornly attachment-dependent, which hampers large-scale viral vaccine production and harvesting.
A group at the National Institute of Diabetes and Digestive and Kidney Diseases at NIH led by Joseph Schloach, Ph.D., has identified a human gene, siat7e, that disrupts attachment of MDCK cells. Expressing the gene in anchorage-dependent MDCK cells tranforms the cells rapidly into suspension cells.
Traditionally, the transformation is achieved in MDCK, CHO, NS0, and other cell lines through serial passage, which is slow. Dr. Schloach’s technique achieves suspension cells at once, at concentrations of just under 1 million cells/mL. MDCK cells that express siat7e not only produce vaccine that is identical to virus that infects attachment-dependent cells, but generate the critical HA1 (hemagglutin-1) antigen at levels 20-fold higher than the parent cells, he says.
Can this technique be applied to short-circuit attachment-dependence of other cell types? “It is possible, but you can never be sure of the results since the transformation is a complex process and depends on several factors,” Dr. Schloach adds.