Solution Studies of Recombinant Human Stromal-Cell-Derived Factor-1

• Published in: Protein expression and purification 2001-04, Vol.21 (3), p.367-377
• Main Authors: Holmes, William D., Consler, Thomas G., Dallas, Walter S., Rocque, Warren J., Willard, Derril H.
• Format: Article
• Language: English
• Subjects: Anti-HIV Agents - chemistry; Anti-HIV Agents - isolation & purification; Anti-HIV Agents - metabolism; chemokine; Chemokine CXCL12; Chemokines, CXC - chemistry; Chemokines, CXC - genetics; Chemokines, CXC - isolation & purification; Chemokines, CXC - metabolism; Chromatography, Gel; Chromatography, High Pressure Liquid; Circular Dichroism; Dimerization; Humans; Light; light scattering; Molecular Weight; Protein Denaturation; Protein Folding; Protein Renaturation; Protein Sorting Signals; Protein Structure, Quaternary; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Scattering, Radiation; SDF-1; sedimentation equilibrium; sedimentation velocity; Sequence Analysis, Protein; Solutions; Thermodynamics; Ultracentrifugation
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2001.1402

Stromal-cell-derived factor-1 (SDF-1α) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1α in Escherichia coli without the leader peptide sequence. The protein was denatured, refolded, and further purified by reversed-phase HPLC. SDF-1α was shown to be >95% pure as judged by SDS-PAGE. The final yield of purified and refolded SDF-1α was 1–2 mg per gram of wet cell paste. The refolded protein is a ligand for the CXCR4 receptor and has been used to block HIV-mediated cell fusion and downmodulates the CXCR4 receptor. Our ability to purify hundreds of milligrams of refolded protein allowed us to conduct detailed studies of the biophysical properties of the protein. We have used a combination of biophysical techniques to study the solution properties of SDF-1α. The average mass of SDF-1α, as determined by static light scattering, gave us the first indications that the chemokine may self-associate. Further investigation with sedimentation velocity ultracentrifugation confirmed the existence of two species. The measured s20, W values defined two masses corresponding to monomer and dimer. Finally, sedimentation equilibrium ultracentrifugation and dynamic light scattering yielded a composite value of 150 ± 30 μM for the dimerization constant. We conclude that SDF-1α exists in a monomer-dimer equilibrium.