title: Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.
abstract: The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.
authors.affiliation: Sandoz Forschungsinstitut Ges.m.b.H., Vienna, Austria.
authors.forename: I
authors.initials: I
authors.lastname: Lindley
authors.forename: H
authors.initials: H
authors.lastname: Aschauer
authors.forename: J M
authors.initials: JM
authors.lastname: Seifert
authors.forename: C
authors.initials: C
authors.lastname: Lam
authors.forename: W
authors.initials: W
authors.lastname: Brunowsky
authors.forename: E
authors.initials: E
authors.lastname: Kownatzki
authors.forename: M
authors.initials: M
authors.lastname: Thelen
authors.forename: P
authors.initials: P
authors.lastname: Peveri
authors.forename: B
authors.initials: B
authors.lastname: Dewald
authors.forename: V
authors.initials: V
authors.lastname: von Tscharner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D002630:Chemotactic Factors / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005813:Genes, Synthetic; D006801:Humans; D016209:Interleukin-8; D008969:Molecular Sequence Data; D009000:Monocytes / Q000502:physiology; D010455:Peptides / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D002630:Chemotactic Factors; D016209:Interleukin-8; D010455:Peptides; D011994:Recombinant Proteins