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Chemists devise inexpensive, benchtop method for marking and selecting cells
Date:1/8/2013

tina tough, sugar-derived material found, for example, in crab shells, squid beaks and the cell walls of fungi. In addition to a main chitin-breaking domain, chitinases have another active structure, a "chitin binding domain" (ChBD). "It makes a super-strong bond with chitin," said Peng. In recent years, scientists have begun to use this high-affinity binding of ChBD and chitin as a marker system, typically for selecting ChBD-tagged proteins in a lab dish. The new method uses ChBD to mark and select cells.

A Powerful Tool

In the basic technique, a new gene can be added to cells within a larger DNA vector that also includes the genetic sequences for ChBD and GFP. The ChBD molecule will be produced in such a way that it ends up being held on the outer surface of its host cell's plasma membraneand the GFP molecule will sit just inside the membrane. The GFP serves as a visual beacon, while the ChBD serves as a handy gripping point for cell selection.

After exposing a culture of test cells to this experimental ChBD-containing vector, the scientists was able to see, via the GFP tags, which cells were expressing them, and was able to select them out easily, with high sensitivity, using magnetic beads coated with chitin. "This is a relatively easy benchtop method," Peng said. Importantly, these selected cells could produce progeny cells that seemed normal and healthy.

Because the ChBD marker, in the vector, is produced in a way that anchors it to a cell's membrane, it also can serve as a powerful tool for selecting just the membrane fraction of a sample of cellular material. Peng and his colleagues demonstrated this using chitin beads to quickly isolate a pure fraction of membrane material from ChBD-marked test cells.

Cellulase enzymes, which break down the ubiquitous plant compound cellulose, also have a high-affinity cellulase-binding domain, which can be employed in the same way as the ChBD.

The scientists exp
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Contact: Mika Ono
mikaono@scripps.edu
858-784-2052
Scripps Research Institute
Source:Eurekalert

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