Synthesis and Expression in Escherichia Coli of the Gene Encoding Monocyte-derived Neutrophil-activating Factor: Biological Equivalence Between Natural and Recombinant Neutrophil-activating Factor

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1988 Dec 1

PMID 3057503

Citations 72

Authors

I Lindley

H Aschauer

J M Seifert

C Lam

W Brunowsky

E Kownatzki

M Thelen

P Peveri

B DEWALD

V Von Tscharner

Affiliations

Soon will be listed here.

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.

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