the respiratory burst, and releasing specific and azurophilic granular contents. Neutrophil-activating factor (NAF) is a peptide produced by human monocytes ...
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 9199-9203, December 1988 Immunology
Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: Biological equivalence between natural and recombinant neutrophil-activating factor (chemotaxis/monokine/cloning)
IVAN LINDLEY*, HEINZ ASCHAUER*, JAN-MARCUS SEIFERT*, CHARLES LAM*, WALTRAUD BRUNOWSKY*, ECKHARD KOWNATZKIt, MARCUS THELENI, PAOLA PEVERIt, BEATRICE DEWALDf, VINZENZ VON TSCHARNERf, ALFRED WALZf, AND MARCO BAGGIOLINIt *Sandoz Forschungsinstitut Ges.m.b.H., Vienna, Austria; tAbteilung Experimentelle Dermatologie, Universitaets-Hautklinik, Freiburg, Federal Republic of Germany; and tTheodor-Kocher-institut, University of Berne, Berne, Switzerland
Communicated by Walter J. Gehring, August 29, 1988 (received for review August 4, 1988)
The neutrophil-activating factor (NAF) puriABSTRACT fied from the conditioned medium of lipopolysaccharidestimulated 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.
Neutrophil-activating factor (NAF) is a peptide produced by human monocytes, which was purified recently (1-3). Independent work in different laboratories has established that peptides of this type are chemotactic for human neutrophils (4-6). We have shown that NAF induces shape changes, the respiratory burst, and exocytosis of specific and azurophilic granules in human neutrophils (7, 8). In potency, NAF compares well with the anaphylatoxin C5a and formylmethionyl peptides (7, 8). NAF appears to act via a selective surface receptor distinct from those of C5a, f-Met-Leu-Phe, platelet-activating factor, and leukotriene B4 (8). Like the known chemotactic peptides, NAF stimulates neutrophils by a mechanism depending on GTP-binding proteins (7), which leads to a transient rise in cytosolic free Ca2+ (8) and to activation of protein kinase C. Studies on NAF have been hampered by the relatively low yield from human monocytes. We therefore decided to produce NAF by recombinant methods and report here the expression of a synthetic NAF gene in Escherichia coli. The recombinant peptide isolated from the bacteria has the same amino acid composition and the same biological activity and potency as natural NAF. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
MATERIALS AND METHODS Purification and Sequencing of Natural NAF. NAF produced by bacterial lipopolysaccharide-stimulated mononuclear leukocytes from human blood was purified (1) and subjected to amino acid-sequence analysis either directly or after chemical modification. SH-aminoethylation was performed with 1 nmol of NAF in 150 ,ul of 0.2 M Tris HCl, pH 8.6/6 M guanidine hydrochloride/2 mM EDTA. The reaction mixture was purged with nitrogen, reduced with 150 nl of tributylphosphine in 10 ,ul of acetonitrile, and aminoethylated with 15 nl of ethylenimine in 15 ,l of acetonitrile in a one-batch process at room temperature (9). After 2 hr, an equal amount of tributylphosphine and ethylenimine was added, and the reaction was continued for another 2 hr under nitrogen. The solution was then acidified with 50% (vol/vol) trifluoroacetic acid to pH 2.0 and desalted by reverse-phase HPLC in 0.1% trifluoroacetic acid with an acetonitrile gradient. SH-methylation was done essentially the same way, except that 260 ,ug of ethyl-4-nitro-benzenesulfonate in 10 ,l of acetonitrile were used instead of ethylenimine (10). Succinylation was obtained by adding 100 ,g of succinic anhydride in 15 ,ul of acetonitrile in three portions within 30 min. The reaction mixture was desalted by reverse-phase HPLC in 50 mM ammonium acetate/ammonia buffer, pH 7.0, with an acetonitrile gradient. Trypsin cleavage of 1 nmol of SHmethylated and aminosuccinylated NAF was done at 37°C for 6 hr in 50 ,l of 0.1 M ammonium acetate/ammonia buffer, pH 8.6, at an enzyme-to-substrate ratio of 1:20 (wt/wt). For carboxyl-terminal analysis, digestion was accomplished with 1 nmol of NAF and 0.1 unit of carboxypeptidase A, 0.01 unit of carboxypeptidase B, or 0.1 unit of carboxypeptidase Y (all from Sigma) according to established methods (11). Cleavage of the Asp-Pro bond of unmodified NAF (1 nmol) was performed in 50 ,ul of 75% (vol/vol) formic acid at 37°C for 48 hr (12). The digestion mixture was then spotted directly on a Polybrene-coated glass filter disc for sequence analysis. Sequencing was done on a model 470 A sequencer (Applied Biosystems, Foster City, CA). Oligonucleotide Synthesis, Purification, and Ligation. The oligonucleotides ON-1 to -6 (Fig. 2) were synthesized on an automated DNA synthesizer (Applied Biosystems 380 A) using standard cyanoethylphosphoamidite chemistry. Deprotected materials were purified by 12% PAGE (13). Full-length oligonucleotides were located by UV-shadowing, excised, electroeluted with a Biotrap BT 100 (Schleicher & Schuell), Abbreviations: NAF, neutrophil-activating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor.
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Proc. Natl. Acad. Sci. USA 85 (1988)
Immunology: Lindley et al.
desalted on a Bio-Gel P2 column in 10%o (vol/vol) ethanol (30 x 0.9 cm, Bio-Rad), and Iyophilized. Samples of ON-1 to -6, 5'-labeled with 32p, were analyzed by PAGE autoradiography and found homogeneous. The sequences were confirmed by the solid-phase Maxam-Gilbert technique (14). For hybridizing and ligation, 250-pmol portions of the oligonucleotides were phosphorylated with 4 gCi of [32P]ATP (5000 ,uCi/mmol; Amersham; 1 Ci = 37 GBq) and 9 units of polynucleotide kinase (Boehringer Mannheim) in 20 1ul of kinase buffer (13) for 40 min at 37TC, followed by a 20-min chase with 5 mmol of unlabeled ATP. The reaction was stopped with 1 1.L of 0.5 M EDTA at 70'C. Phosphorylated oligonucleotides were purified on Nensorb 20 cartridges (DuPont) according to the manufacturer's instructions, and the yield was =90%. Simultaneous hybridizing and ligation of 30-100 pmol of 5'-phosphorylated ON-2 to -5 and unphosphorylated ON-1 and ON-6 was done in 25 Al of 125 mM Tris'HCI, pH 7.6, containing 25 mM MgCl2. The mixture was heated to 90°C for 4 min, cooled to 14°C within 3 hr, and incubated for another 14 hr at 14°C. The volume was adjusted to 60 ,ul with 50 mM Tris HCI, pH 7.6/10 mM MgCl2/10 mM dithiothreitol/1 mM ATP/bovine serum albumin at 0.1 mg/ml containing 1800 units of T4 ligase (New England Biolabs). Ligation was performed for 14 hr at 14°C and stopped by addition of 1 ul of 0.5 M EDTA and heating to 70°C. After phenol extraction and ethanol precipitation (13) the products were purified by 8% PAGE/7 M urea, excised, electroeluted, and ethanol precipitated, yielding finally 5-
8%. Cloning of Synthetic Gene. A sample of 110 ng of the isolated synthetic NAF gene was ligated with 260 ng of agarose gel-purified Sph I/BamHI-cut pBS M13- plasmid DNA (Stratagene, La Jolla, CA). One-tenth of this ligation mixture was used to transform 200 ul of E. coli strain 5K cells, made competent by the method of Hanahan (15), and -320 ampicillin-resistant colonies were produced per ng of input DNA. Six clones were picked and grown overnight in L broth/ampicillin (Difco). Plasmid DNA was prepared (16) and run on a 1% agarose gel, and pure supercoiled DNA was isolated by electrophoresing the bands into 3MM paper (Whatman) and centrifuging into Eppendorf tubes. After phenol extraction and ethanol precipitation, the DNA was sequenced by the alkali-denatured plasmid chain-termination method (17), using T3 and T7 primers (Stratagene) and the sequenase enzyme (United States Biochemical, Cleveland). The E. coli expression vector pIL402(Term) contains a unit comprising the E. coli tryptophan promoter (18), the human granulocyte/macrophage colony-stimulating factor (GMCSF) gene, and a synthetic transcription terminator CCCGGGCGATTCATCGCCCGGG inserted between the EcoRI and Sal I sites of pAT153 (19). Plasmid DNA was cut with Cla I and BamHI to excise the GM-CSF gene, and the vector fragment was isolated from an agarose gel and ligated to a similarly isolated 237-base-pair (bp) Cla I/BamHI fragment containing the NAF gene from one of the correct clones (clone 6). The resulting expression plasmid, designated p(NAF)-6T3 (Fig. 3A), was transformed into E. coli cells of strain HB101. Expression. Fermentation of E. coli containing p(NAF)6T3 was performed at 37°C in a 35-liter working volume of M9 medium (20) containing 0.2% glucose, 0.5% Casamino acids (Difco), tryptophan at S mg/liter and ampicillin at 100 ,ug/ml. When OD6w reached 2.8, indole acrylic acid (Sigma) in ethanol was added to a final concentration of 20 mg/liter. After 4 hr the OD6w had increased to 13.5, and centrifugation yielded a 516-g cell pellet. Purification of Recombinant NAF. E. coli cells containing recombinant NAF were stored at -20TC. Batches of 100 g were washed with 100 ml of 20 mM Tris HCl, pH 8.0/50 mM NaCl, resuspended in 250 ml of the same buffer, and
disrupted at 2500 psi (1 psi = 6.89 kPa) in a French press (Aminco). After centrifugation at 47,000 x g for 40 min the pellet was resuspended in the same buffer, recentrifuged, and stored at -20'C. Portions of 10 g were suspended in 100 ml of 50 mM Mes-NaOH, pH 6.5/6 M guanidine hydrochloride, stirred for 1 hr, and dialyzed against 0.5% acetic acid. The dialysate was clarified by centrifugation and loaded in aliquots on a Mono S column (HR 5/5; Pharmacia). The column was washed with 50 mM Mes-NaOH, pH 6.5/0.2 M NaCl and eluted at a rate of0.5 ml/min with a linear gradient in the same buffer (0.2-0.5 M NaCl). The fractions with the highest NAF content were pooled and chromatographed on a wide-pore reverse-phase HPLC column (0.46 x 125 mm Vydac C4 TP) in 0.1% trifluoroacetic acid and a 20-60% acetonitrile gradient at a flow rate of 1 ml/min. Electrophoresis and Western Blots. SDS/PAGE was performed as described (1). Gels were silver-stained or blotted onto nitrocellulose at pH 10.4. Blots were developed with a mouse anti-NAF antiserum. Antibodies against NAF were raised in mice with an =80% pure preparation of natural NAF bound to phosphocellulose. Fractions obtained during purification were analyzed for the presence of NAF on precast 20% SDS gels under reducing conditions using a Phast system electrophoresis apparatus (Pharmacia). A 1
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10
5
Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-
T 7-26 --------.------------------
20
25
30
Pro-Phe-His-Pro-Lys-Phe-Ile-Lys-Glu-Leu-Arg-Val-Ile-Glu-Ser------------------------------------------>
35
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40
45
Gly-Pro-His-Cys-Ala-Asn-Thr-Glu-Ile-Ile-Val-Lys-Leu-Ser-Asp-
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T 27-47
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50 60 55 Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg------>
65
70
72
Val-Val-Glu-Lys-Phe-Leu-Lys-Arg-Ala-Glu-Asn-Ser 61-72 -------------------. ------------------------------.
