On-column refolding of recombinant chemokines for NMR studies and biological assays.
We have applied an efficient solid-phase protein refolding method to the milligram scale production of natively folded recombinant chemokine proteins. Chemokines are intensely studied proteins because of their roles in immune system regulation, response to inflammation, fetal development, and numerous disease states including, but not limited to, HIV-1/AIDS, cancer metastasis, Crohn's disease, asthma and arthritis. Many investigators use recombinant chemokines for research purposes, however these proteins partition almost exclusively to the inclusion body fraction when produced in Escherichia coli. A major hurdle is to correctly refold the chemokine and oxidize the two highly conserved disulfide bonds found in nearly all chemokines. Conventional methods for oxidation and refolding by dialysis or extreme dilution are effective but slow and yield large volumes of dilute chemokine. Here we use an on-column approach for rapid refolding and oxidation of four chemokines, CXCL12/SDF-1alpha (stromal cell-derived factor-1alpha), CCL5/RANTES, XCL1/lymphotactin, and CX3CL1/fractalkine. NMR spectra of SDF-1alpha, RANTES, lymphotactin, and fractalkine indicate these chemokines adopt native structures. On-column refolded SDF-1alpha is fully active in an intracellular calcium flux assay. Our success with multiple SDF-1alpha mutants and members of all four chemokine subfamilies suggests that on-column refolding is a robust method for preparative-scale production of recombinant chemokine proteins.
Veldkamp
Christopher T
CT
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Peterson
Francis C
FC
Hayes
Paulette L
PL
Mattmiller
Jessie E
JE
Haugner
John C
JC
de la Cruz
Norberto
N
Volkman
Brian F
BF
D018925:Chemokines / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003956:Dialysis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018925:Chemokines; D011994:Recombinant Proteins