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May 2009

Designer Protein Grabs Cover

Stable affinity reagent developed as antibody alternative

Portrait of Curt Boschek
Curt Boschek
Journal cover  PEDS
The modeled structure of Top7CB1 is displayed as a cartoon with sheets in red, helices in blue, the 10 best-scored conformations of the inserted CB1 sequence in yellow, and connecting regions in green. At the bottom is the Top7CB1 sequence following the same color scheme with the addition of grey for portions that are excluded from the structure. Enlarged View

Results: Move over, Tommy Hilfiger. Top7CB1, designed by researchers at Pacific Northwest National Laboratory, is grabbing the spotlight. This synthetic protein can specifically bind a protein targeted by the human immunodeficiency virus, or HIV, that can lead to AIDS. Top7CB1 can also be used as an inexpensive and effective alternative to antibodies. The work is the cover story in the May 2009 issue of Protein Engineering Design & Selection.

Why it matters: Antibodies are one of the weapons used to fight disease or detect harmful substances. These proteins are one of the most commonly used reagents in the laboratory because they can be used to bind, recognize and quantify specific targets, such as toxins or proteins that specify different disease states.

Antibodies can be generated against virtually any target by immunization or by in vitro selection. However, they are large and frequently unstable, which makes them difficult to use for many practical applications.  As a result, scientists are working to engineer smaller antibody fragments that remain specific and stable while being easy to produce. But again, designing these engineered fragments to be structurally robust while retaining their binding specificity has been difficult.

An emerging alternative is the use of intrinsically stable proteins as scaffolds for the generation of novel binding agents instead of generating natural or engineered antibodies. In fact, these novel scaffolds could be used in adverse environments such as those found in parts of the human body or in industry, where antibodies fail.

The aim of the PNNL study was to design a highly stable affinity reagent—a specific binding molecule—based on the synthetic protein Top7 to assess its viability as a general affinity scaffold. Top7 is a small protein computationally designed by University of Washington scientists to be extremely stable. Its small size, known structure and very stable configuration make it an ideal scaffold for an affinity reagent.

Methods: The researchers selected a site in Top7 to insert CB1, a peptide constructed from a well-characterized peptide, PDP-CB1, that comes from a region of an anti-CD4 antibody. CD4 is a protein on the surface of immune cells that helps protect against infections such as HIV. Inserting this peptide resulted in the variant called Top7CB1. Team members then evaluated the structural effect of the variant using molecular dynamics simulations that suggested that Top7CB1 retains conformational stability at temperatures greater than 100°C—hotter than boiling water.

The modified Top7 also bound CD4 and, consistent with simulations, was extremely resistant to thermal and chemical structural change—retaining its secondary structure up to at least 95°C. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent that could serve as a robust and inexpensive alternative to antibodies.

What's next: The PNNL team is currently using complementary determining region loops harvested from antibody libraries at PNNL as diversity elements for building libraries of Top7 variants for selection through yeast surface display. Based on suggestions that the destabilization effects of multiple loop addition are not synergistic, they are also exploring the use of multiple binding sequences at other regions within Top7 to further increase specificity and affinity.

Acknowledgments: The research team includes Curt Boschek (lead author), Thereza Soares, Heather Engelmann, Tjerk Straatsma and Cheryl Baird, all PNNL: and former PNNL staff David Apiyo and Noah Pefaur. This work was supported by the U.S. Department of Energy with Laboratory Directed Research and Development funding through the Biomolecular Systems Initiative. A portion of this research was performed using EMSL, a national scientific user facility sponsored by DOE's Office of Biological and Environmental Research located at PNNL.

Reference: Boschek CB, DO Apiyo, TA Soares, HE Engelmann, N Pefaur, TP Straatsma, and CL Baird.  2009.  "Engineering an ultra-stable affinity reagent based on Top7."  Protein Engineering, Design & Selection 22(5):1-8, doi:10.1093/protein/gzp007.

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