Only a fraction of proteins can be overproduced in E. coli in sufficient yield and without the formation of inclusion-body aggregates or the proteolytic degradation of expressed proteins. Alternative expression systems include cell cultures from eukaryotic organisms, such as insect cells, and cell-free, in vitro protein expression.

The latter is the focus of John Markley's group at the Center for Eukaryotic Structural Genomics at the University of Wisconsin, Madison. Collaborating with Ehime University and CellFree Sciences, both in Japan, Markley and colleagues developed a pipeline using wheat germ cell-free protein translation as a way to produce proteins for nuclear magnetic resonance (NMR) structure determination.

Credit: R. DAVIES

Cell-free systems simplify the purification efforts, as “only the protein of interest is expressed and labelled, thus the background is cleaner”, says Markley (pictured). For the NMR studies, his group has been getting twice as many folded proteins using the cell-free system than with expression in E. coli. The cell-free method also requires smaller volumes, avoiding lengthy concentration steps, and it lends itself to the labelling of proteins, which is a requirement for NMR. “But there is a steep learning curve for learning how to do a cell-free system,” says Dmitriy Vinarov, who is responsible for high-throughput production at the centre. One downside of cell-free systems is the expense of the reagents, especially when success rates are not high. “About 79% of human proteins will be expressed, about one half of those will produce protein in sufficient quantities for structural studies, and half of those will remain stably folded for NMR studies,” says Markley.

Cell-free and cell-based systems are not mutually exclusive. “Each has unique advantages and capabilities so we will continue to do both,” explains Markley. By using a new cloning system from Promega the researchers have been able to transfer target cDNAs from cell-free to cell-based expression systems to achieve greater flexibility. “We still have a lot to learn. It is working quite well for us but we still have improvements to make,” says Vinarov. “The technology is not quite primetime.”

Cell-free systems are available from companies such as Roche Applied Science, QIAGEN and Invitrogen, as well as from CellFree Sciences.

L.B.