Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.
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.
Lindley
I
I
Sandoz Forschungsinstitut Ges.m.b.H., Vienna, Austria.
Aschauer
H
H
Seifert
J M
JM
Lam
C
C
Brunowsky
W
W
Kownatzki
E
E
Thelen
M
M
Peveri
P
P
Dewald
B
B
von Tscharner
V
V
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
D002630:Chemotactic Factors; D016209:Interleukin-8; D010455:Peptides; D011994:Recombinant Proteins