title: Synthesis and expression in Escherichia coli of a human neutrophil activating protein-1/interleukin-8 gene.
abstract: The complete gene coding for human neutrophil activating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the PRPL tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to > 95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of < 10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP-1/IL-8 was determined using the Edman method and was shown to be identical to that of the native protein.
authors.affiliation: National Laboratory of Molecular Virology & Genetic Engineering, Chinese Academy of Preventive Medicine, Beijing.
authors.forename: D Y
authors.initials: DY
authors.lastname: Jin
authors.forename: R H
authors.initials: RH
authors.lastname: Xu
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: Y D
authors.initials: YD
authors.lastname: Hou
mesh_terms: D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011817:Rabbits
chemical_list: D016209:Interleukin-8; D004247:DNA