title: Expression of recombinant human interleukin-8 and its purification using a single buffer system.
abstract: Chemokines, a class of small secreted proteins, direct immune cells to their target sites and play an important role in chronic inflammations and allergies. To study their interactions with their cellular receptors or potential inhibitors large quantities of chemokines are required. Here we present a fast and efficient strategy to purify the human chemokine interleukin-8 (IL-8, CXCL8). The chemokine is expressed with a pelB-leader peptide that is cleaved off its N-terminus by an endogenous bacterial peptidase. This yields wild-type 72aa IL-8 with a serine at its N-terminus. IL-8 is recovered in the soluble fraction after lysis while pelB-IL8 fusion protein remains in the pellet. Interleukin-8 is purified via cation exchange chromatography and heparin affinity chromatography using a single inexpensive buffer system. No dialysis or membrane filtration steps are required and the final protein fractions may be used without any desalting steps. The use of 0.5% Triton X-114 in the lysis buffer leads to low endotoxin levels in the resulting protein. The protein can be eluted from the gel filtration column with a variety of buffers and is ready to be used in binding assays and activity assays.
authors.affiliation: Karlsruhe Institute of Technology, Institute of Organic Chemistry, Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany. dana.wiese@ifg.fzk.de
authors.forename: Dana
authors.initials: D
authors.lastname: Wiese
authors.forename: Katja
authors.initials: K
authors.lastname: Schmitz
mesh_terms: D002021:Buffers; D002846:Chromatography, Affinity; D003362:Cost-Benefit Analysis; D004731:Endotoxins / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002021:Buffers; D004731:Endotoxins; D016209:Interleukin-8; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40