title: Production of Recombinant Chemokines and Validation of Refolding.
abstract: The diverse roles of chemokines in normal immune function and many human diseases have motivated numerous investigations into the structure and function of this family of proteins. Recombinant chemokines are often used to study how chemokines coordinate the trafficking of immune cells in various biological contexts. A reliable source of biologically active protein is vital for any in vitro or in vivo functional analysis. In this chapter, we describe a general method for the production of recombinant chemokines and robust techniques for efficient refolding that ensure consistently high biological activity. Considerations for initiating development of protocols consistent with Current Good Manufacturing Practices (cGMPs) to produce biologically active chemokines suitable for use in clinical trials are also discussed.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA; Department of Chemistry, University of Wisconsin-Whitewater, Whitewater, Wisconsin, USA. Electronic address: veldkamc@uww.edu.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Veldkamp
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Koplinski
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Davin R
authors.initials: DR
authors.lastname: Jensen
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA; The Eppley Institute and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors.forename: Kaitlin M
authors.initials: KM
authors.lastname: Smits
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA; The Eppley Institute and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors.forename: Brittney L
authors.initials: BL
authors.lastname: Smith
authors.affiliation: Biological Process Development Facility, College of Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Scott K
authors.initials: SK
authors.lastname: Johnson
authors.affiliation: Department of Pediatrics, Children's Hospital and Medical Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Lettieri
authors.affiliation: Biological Process Development Facility, College of Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Wallace G
authors.initials: WG
authors.lastname: Buchholz
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA; The Eppley Institute and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.
authors.forename: Joyce C
authors.initials: JC
authors.lastname: Solheim
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
mesh_terms: D002633:Chemotaxis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006152:Cyclic GMP / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D006152:Cyclic GMP
keywords: Chemokine; Chemokine oxidation; Chemokine purification; Chemokine refolding; Expression; Mass spectrometry; Mass spectrometry of chemokines; NMR; Protein; Protein NMR; Protein folding; Purification; Recombinant chemokines; Recombinant cytokines; Refolding; cGMP