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Solution studies of recombinant human stromal-cell-derived factor-1.
W D Holmes1, T G Consler, W S Dallas
1Department of Molecular Sciences, GlaxoWellcome Research and Development, Research Triangle Park, NC 27709, USA. wdh17912@gsk.com
Protein Expression and Purification
|April 3, 2001
Summary
Stromal-cell-derived factor-1 (SDF-1alpha), a key chemokine, was successfully produced and purified. Biophysical studies revealed that SDF-1alpha exists in a monomer-dimer equilibrium in solution.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine.
- It is constitutively expressed in bone-marrow-derived stromal cells.
- SDF-1alpha is a ligand for the CXCR4 receptor and plays a role in HIV-mediated cell fusion.
Purpose of the Study:
- To produce recombinant SDF-1alpha.
- To characterize its biophysical properties, specifically its solution behavior.
- To investigate potential self-association of SDF-1alpha.
Main Methods:
- Recombinant expression of SDF-1alpha in Escherichia coli.
- Denaturation, refolding, and purification using reversed-phase HPLC.
- SDS-PAGE for purity assessment.
- Static light scattering, sedimentation velocity ultracentrifugation, sedimentation equilibrium ultracentrifugation, and dynamic light scattering for biophysical characterization.
Main Results:
- Recombinant SDF-1alpha was produced with >95% purity.
- Yield of purified protein was 1-2 mg per gram of wet cell paste.
- Biophysical studies indicated that SDF-1alpha self-associates.
- Evidence for monomer and dimer species was confirmed.
- A dimerization constant of 150 +/- 30 microM was determined.
Conclusions:
- SDF-1alpha can be produced recombinantly in a functional form.
- SDF-1alpha exists in a monomer-dimer equilibrium in solution.
- Understanding this equilibrium is crucial for its biological function and therapeutic applications.