title: Rapid site-directed mutagenesis of chemokines and their purification from a bacterial expression system.
abstract: Chemokines are a family of small chemoattractant cytokines implicated in the recruitment and migration of leukocytes from the blood into tissues during disease and routine immune homeostasis. Although there are many similarities in the structure and function of certain chemokines, the importance of many residues in the function of these proteins is yet to be determined, and studies from related chemokines have shown that similar sequences may play different roles in each protein. The migration-inducing capacity of many chemokines is thought to involve the cell surface glycosaminoglycan (GAG), heparan sulphate (HS), which may assist in the formation of an immobilised chemokine gradient within inflamed tissues. To examine the heparan sulphate binding ability of the CC chemokine monocyte chemoattractant protein (MCP)-3 and its importance in chemotactic migration, we have identified and mutated conserved basic residues within the mature MCP-3 protein to the neutral amino acid alanine using a novel inverse polymerase chain reaction (I-PCR) method that rapidly generates essentially 100% mutational efficiency due to decreased requirements for template DNA and an alkaline denaturation step; this increased mutational efficiency reduces both screening time and sequencing costs. We also describe an optimised method for the expression of soluble, correctly folded MCP-3 in a bacterial system using nickel affinity columns and reverse-phase fast protein liquid chromatography (RP-FPLC), and achieve purified yields of up to 0.4 mg/l of initial culture medium after 5 h of induction. These optimised methods could work equally well for any small circular plasmid (< or =4.5 kb) incorporating a polyhistidine tag.
authors.affiliation: Department of Surgery, Medical School, University of Newcastle-upon-Tyne, Framlington Place, NE2 4HH Newcastle-upon-Tyne, UK.
authors.forename: Julie H
authors.initials: JH
authors.lastname: Wain
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Palmer
authors.forename: John A
authors.initials: JA
authors.lastname: Kirby
authors.forename: Simi
authors.initials: S
authors.lastname: Ali
mesh_terms: D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D054410:Chemokine CCL7; D018925:Chemokines / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D016207:Cytokines; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation
chemical_list: C515450:CCL7 protein, human; D054410:Chemokine CCL7; D018925:Chemokines; D016207:Cytokines; D008070:Lipopolysaccharides; D018945:Monocyte Chemoattractant Proteins; D002118:Calcium