title: Optimized procedures for producing biologically active chemokines.
abstract: We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.
authors.affiliation: GlaxoSmithKline, Biological Reagents & Assay Development, Mail Code: UE0548, 709 Swedeland Road, King of Prussia, PA 19406, USA. Quinn.2.Lu@gsk.com
authors.forename: Quinn
authors.initials: Q
authors.lastname: Lu
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Burns
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: McDevitt
authors.forename: Taylor L
authors.initials: TL
authors.lastname: Graham
authors.forename: Abby J
authors.initials: AJ
authors.lastname: Sukman
authors.forename: James A
authors.initials: JA
authors.lastname: Fornwald
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Tang
authors.forename: Kathleen T
authors.initials: KT
authors.lastname: Gallagher
authors.forename: Gerald E
authors.initials: GE
authors.lastname: Hunsberger
authors.forename: James J
authors.initials: JJ
authors.lastname: Foley
authors.forename: Dulcie B
authors.initials: DB
authors.lastname: Schmidt
authors.forename: John J
authors.initials: JJ
authors.lastname: Kerrigan
authors.forename: Tia S
authors.initials: TS
authors.lastname: Lewis
authors.forename: Robert S
authors.initials: RS
authors.lastname: Ames
authors.forename: Kyung O
authors.initials: KO
authors.lastname: Johanson
mesh_terms: D018925:Chemokines / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018925:Chemokines; D011993:Recombinant Fusion Proteins