Faculty Collaboration Database

On-column refolding of recombinant chemokines for NMR studies and biological assays. Protein Expr Purif 2007 Mar;52(1):202-9

Date

10/31/2006

Pubmed ID

17071104

Pubmed Central ID

PMC1868460

DOI

10.1016/j.pep.2006.09.009

Scopus ID

2-s2.0-33845933655 (requires institutional sign-in at Scopus site)   32 Citations

Abstract

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.

Author List

Veldkamp CT, Peterson FC, Hayes PL, Mattmiller JE, Haugner JC 3rd, de la Cruz N, Volkman BF

Authors

Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Chemokines
Cloning, Molecular
Dialysis
Escherichia coli
Humans
Kinetics
Magnetic Resonance Spectroscopy
Plasmids
Protein Folding
Recombinant Proteins
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization