title: High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column.
abstract: We developed a highly efficient expression system for the production of interleukin-8 (IL-8) in Escherichia coli. A synthetic gene used in the vector was designed to code for the 72-amino-acid form of IL-8 and incorporate additional new restriction sites. IL-8 was expressed in very large amounts in the periplasmic space and extracted by a gentle method which did not utilize denaturants. About 69% of the protein extracted from the periplasmic space was properly processed IL-8. A single anti-IL-8 monoclonal antibody affinity chromatography column yielded homogeneous IL-8 as determined by HPLC molecular sieve chromatography and amino-terminal sequencing. Between 14 and 22 mg of IL-8 was purified per liter of bacterial culture, in which the wet weight of E. coli was 7.6 g/liter. The recombinant IL-8 was fully active compared to published data and a commercially available preparation of recombinant IL-8. Our IL-8 and the commercial product had identical neutrophil binding isotherms, chemotactic activities, and enzyme release properties.
authors.affiliation: Department of Biochemistry, University of Texas Health Center, Tyler 75710, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Miller
authors.forename: A B
authors.initials: AB
authors.lastname: Cohen
authors.forename: F K
authors.initials: FK
authors.lastname: Carr
authors.forename: S
authors.initials: S
authors.lastname: Hayashi
authors.forename: C Y
authors.initials: CY
authors.lastname: Chiu
authors.forename: C T
authors.initials: CT
authors.lastname: Lee-Ng
authors.forename: G
authors.initials: G
authors.lastname: Mullenbach
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D002634:Chemotaxis, Leukocyte; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D016209:Interleukin-8 / Q000031:analogs & derivatives / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D017421:Sequence Analysis
chemical_list: D016209:Interleukin-8; D011994:Recombinant Proteins