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Recombinant decorsin: dynamics of the RGD recognition site
A M Krezel1, J S Ulmer, G Wagner
1Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA. Andrzej.M.Krezel@vanderbilt.edu
Protein Science : a Publication of the Protein Society
|September 7, 2000
Summary
Recombinant decorsin, a potent platelet aggregation inhibitor, was successfully produced in E. coli. Its structure and function were confirmed to be identical to native decorsin, with a restricted RGD site conformation.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Decorsin is a potent inhibitor of platelet aggregation, acting as an antagonist to integrin alphaIIbbeta3.
- Decorsin was originally isolated from the leech Macrobdella decora.
Purpose of the Study:
- To design, construct, and express a synthetic gene encoding decorsin in Escherichia coli.
- To characterize the purified recombinant decorsin and compare it to the native protein.
- To elucidate the structural and dynamic properties of decorsin, particularly its RGD adhesion site.
Main Methods:
- Gene synthesis and expression in E. coli using stII signal sequence and alkaline phosphatase promoter.
- Protein purification via size-exclusion filtration and reversed-phase high-performance liquid chromatography.
- Characterization using amino acid composition, mass spectral analysis, biological activity assays, and Nuclear Magnetic Resonance (NMR) spectroscopy.
Main Results:
- Purified recombinant decorsin was indistinguishable from leech-derived decorsin in composition, mass, and biological activity.
- Complete sequential and stereospecific assignments of NMR resonances were established.
- The RGD adhesion site is located at the apex of an exposed hairpin loop, exhibiting restricted conformation despite overall loop flexibility.
Conclusions:
- Recombinant production of functional decorsin in E. coli is feasible.
- NMR analysis reveals unique structural and dynamic features of the decorsin RGD site.
- Decorsin's restricted RGD conformation may contribute to its potent integrin alphaIIbbeta3 antagonism and platelet aggregation inhibition.