Huimin Zhao is
a professor in the Department of Chemical and Biomolecular Engineering and member of the Institute for Genomic Biology at
Illinois.
The new technique, published online
ahead of regular publication by the Proceedings of the National
Academy of Sciences, combines the advantages of directed evolution
and computationally driven rational design, said Huimin Zhao, a
professor in the
Department of Chemical and Biomolecular Engineering and member
of the Institute for Genomic
Biology at the U of I.
Zhao's team, using yeast and mammalian cells, altered the
specificity of human estrogen receptor alpha by 100 million times so
it would bind preferentially to a nontoxic synthetic molecule
(4,4'-dihydroxybenzil) over the natural estrogen 17-beta-estradiol.
Such selectivity moves researchers
closer to designing synthetic molecules that will attach only to
targeted receptors to activate or deactivate desired gene expression
in living systems, which could lead to advances in such applications
as gene therapy, metabolic engineering, functional genomics, enzyme
engineering and animal disease model studies.
Many previous attempts, using a
variety of molecular methods, have involved time-consuming
approaches that have resulted in unintended activity when
nontargeted receptors have responded to the new molecules.
"I'm not saying that we have solved
the problem, but we have shown that our approach can be very
efficient and done successfully," said Zhao, also an affiliate in
the departments of Chemistry
and Bioengineering and
member of the Center for Biophysics and
Computational Biology. "We were able to alter the ligand
(molecule) selectively by 10 to the eighth in mammalian cells. No
one has had this high level of success."
The Illinois approach, Zhao said, is
more general, quicker to accomplish and more accurate than a
scientifically hailed combinational approach published in
Proceedings of the National Academy of Sciences last October by
researchers at the Georgia Institute of Technology. In their paper,
the Georgia scientists used random mutagenesis and chemical
complementation to develop a yeast-based system that made a retinoid
X receptor, a nuclear hormone receptor, recognize and bind to a
synthetic molecule.
[to top of second column in this article] |
 The protein-engineering approach
used by Zhao's team used directed evolution, which mimics natural
evolution in a test tube, to force rapid evolution of human estrogen
receptor with new ligand specificity. This process is done mainly
through stepwise, site-saturation mutagenesis and high throughput
screening. The sites of human estrogen receptor chosen for
saturation mutagenesis were identified through rational design,
which involves computational modeling and biochemical and genetic
studies to predict the interactions between the receptor and the
ligand and the myriad molecular interactions that take place to
drive gene expression. The engineered genetic changes subsequently
make the receptor highly sensitive to the synthetic molecule that is
introduced.
"We envision that the described
technology could provide a powerful, broadly applicable tool for
engineering receptors/enzymes with improved or novel ligand/substrate
specificity," Zhao said.
Co-authors with Zhao were Karuppiah
Chockalingam and Zhilei Chen, both doctoral students in chemical and
biomolecular engineering, and John A. Katzenellenbogen, a Swanlund
Endowed Chair in chemistry and affiliate of the
Beckman Institute of Advanced
Science and Technology at the U of I.
A patent is being sought for the
protein-engineering technology and gene switch.
A National Science Foundation
Faculty Early Career Development Award grant to Zhao funded the
research.
[Provided by Jim Barlow, life sciences editor,
University of Illinois at
Urbana-Champaign]
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