A new method developed by Cornell
biological engineers offers an efficient way to make proteins for use in medicine
or industry without the use of live cells. The proteins made in this way include
many that cannot be produced by current biotechnology.
 | | Pads of P-gel, each about 1 millimeter square, in which DNA has been embedded to code for a fluorescent compound. |
Current methods employ vats of genetically modified bacteria or mammalian cells
that churn out proteins for such pharmaceuticals as insulin or human growth
hormone. But there are many proteins that bacteria or cells cannot tolerate.
Anti-microbials, for example, are meant to kill bacteria and so would kill the
host. And many key proteins that are important in regulating the normal life
of a cell would also kill the host if overproduced inside a cell.
Researchers have tried mixing DNA that codes for the desired protein with
the amino acids from which proteins are made along with ribosomes (cell structures
that assemble proteins) and other helper chemicals in a test tube. Cornell's
faster, more efficient process weaves the coding DNA into an artificial gel
made of synthetic DNA.
The process is described in the March 29 online edition of the journal Nature
Materials by Dan Luo, Cornell associate professor of biological and environmental
engineering, and colleagues, and will appear in a forthcoming print edition.
Luo's group has pioneered the use of synthetic DNA as a self-assembling construction
material. Strands of DNA that are designed to be complementary over a small
part of their length can join together into various shapes. In this application
they form crosses, which in turn link at their ends to form a 3-D matrix. This
makes a hydrogel, a spongy material that absorbs water without dissolving in
the water.
To make a protein-producing gel, which Luo calls a P-gel, the synthetic DNA
is also made to include sequences that join to the ends of plasmids -- strands
of DNA that code for the desired protein. A mix of X-shaped and plasmid DNA
then assembles into a gel with genes coding for the desired protein integrated
throughout. To increase the surface area for reaction, tiny drops of the P-gel
are molded into pads about 1 millimeter square by 20 microns (millionths of
a meter) thick. Several hundred pads are then placed in a solution of amino
acids and protein-making machinery extracted from living cells.
The result, Luo reports, is to produce proteins up to 300 times more efficiently
than when the same reactions are carried out with DNA floating freely in the
same solution. The system has so far been tested with 16 proteins, including
several that are toxic or would otherwise be impossible to make in living cells.
Workers in Luo's lab have spent nearly a year trying variations of the process
to find out why it works so well and suggest several reasons: Genes locked into
the hydrogel are protected from damage they might suffer when floating free;
much more DNA can be packed into the P-gels than can be dissolved in a given
amount of solution; and because the genes are close together, enzymes taking
part in the transcription process remain close by and can perform more quickly.
Luo, Alan Biloski, Ph.D. '82, a senior lecturer in the Johnson School, and
two other partners have formed DNANO, a company to explore commercial applications
of the P-gel process and other applications of DNA materials.
The research is supported by the National Science Foundation, the U.S. Department
of Agriculture and the New York State Foundation for Science, Technology and
Innovation.
Posted April 1st, 2009
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