Solution studies of recombinant human stromal-cell-derived factor-1.
Stromal-cell-derived factor-1 (SDF-1alpha) 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-1alpha in Escherichia coli without the leader peptide sequence. The protein was denatured, refolded, and further purified by reversed-phase HPLC. SDF-1alpha was shown to be >95% pure as judged by SDS-PAGE. The final yield of purified and refolded SDF-1alpha 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-1alpha. The average mass of SDF-1alpha, 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 s(20, 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 microM for the dimerization constant. We conclude that SDF-1alpha exists in a monomer-dimer equilibrium.
Department of Molecular Sciences, GlaxoWellcome Research and Development, Research Triangle Park, NC 27709, USA. wdh17912@gsk.com
W D
WD
Holmes
T G
TG
Consler
W S
WS
Dallas
W J
WJ
Rocque
D H
DH
Willard
D019380:Anti-HIV Agents / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D006801:Humans; D008027:Light; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D021382:Protein Sorting Signals; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D020539:Sequence Analysis, Protein; D012996:Solutions; D013816:Thermodynamics; D014461:Ultracentrifugation
D019380:Anti-HIV Agents; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012996:Solutions