Synthesis and Expression in Escherichia coli of the Gene Encoding Monocyte-Derived Neutrophil-Activating Factor: Biological Equivalence between Natural and Recombinant Neutrophil-Activating Factor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 23; pp. 9199 - 9203
• Main Authors: Lindley, Ivan, Aschauer, Heinz, Seifert, Jan-Marcus, Lam, Charles, Brunowsky, Waltraud, Kownatzki, Eckhard, Thelen, Marcus, Peveri, Paola, Dewald, Beatrice, Von Tscharner, Vinzenz, Walz, Alfred, Baggiolini, Marco
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.12.1988; National Acad Sciences
• Subjects: Amino Acid Sequence; Amino acids; Base Sequence; Biological and medical sciences; Biological Assay; Chemotactic Factors - genetics; Cloning, Molecular; Complementary DNA; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gels; Gene expression; Generally accepted auditing standards; Genes; Genes, Synthetic; Humans; Immunology; Interleukin-8; Ligation; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Monocytes - physiology; Neutrophils; Oligonucleotides; Peptides - genetics; Peptides - physiology; Plasmids; Recombinant Proteins - pharmacology; Gene expression
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.23.9199

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: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS. 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.