May 26, 1987 - (Whittemore et al., in press) and is similar to the chicken. NGF construct in all regards except for the absence of a polyadenylation signal and a ...
Vol. 8, No. 1
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1988, p. 452-456
027Q-7306/88/010452-05$02.00/0 Copyright C) 1988, American Society for Microbiology
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Production and Characterization of Biologically Active Recombinant Beta Nerve Growth Factor FINN HALLBOOK,lt* TED EBENDAL,2 AND HAKAN PERSSON1t Department of Medical Genetics, The Biomedical Center, Uppsala University, S-75123 Uppsala,1 and Department of Zoology, Uppsala University, S-75122 Uppsala Sweden ,
Received 26 May 1987/Accepted 24 September 1987
DNA fragments encoding either rat or chicken beta nerve growth factor (NGF) were inserted in the expression vector p91023(B) for transient expression in COS cells. The two NGF constructs produced RNA transcripts and proteins of the predicted sizes. Conditioned media from the transfected cells stimulated neurite outgrowth from cultured chicken embryo sympathetic ganglia. The results show that the rat or chicken NGF gene can direct the synthesis of a biologically active NGF protein after transfection of COS cells.
ation signal, ATTAAA (14), are present in the inserted fragment. A construct with the chicken NGF fragment inserted in the opposite orientation was also isolated and used as a negative control. The rat NGF construct (pERN; Fig. 1) contains a 771-bp BstEIl-PstI fragment from the 3' exon of the rat NGF gene (Whittemore et al., in press) and is similar to the chicken NGF construct in all regards except for the absence of a polyadenylation signal and a 3' untranslated region. The lack of a polyadenylation signal in the inserted rat NGF fragment is compensated for by the presence of a corresponding sequence in the p91023(B) vector (Fig. 1) (8, 23). Total RNA was prepared from COS cells (4) transfected with the NGF constructs by a DEAE-dextran--chloroquine transfection protocol (12). The RNA was separated on an agarose gel, blotted onto a nitrocellulose filter, and hybridized to nick-translated NGF probes. This analysis revealed transcripts with sizes corresponding to the length predicted from the structure of the constructs. pECN transfections produced two transcripts of 1.2 kilobase pairs (kb) and 2.2 kb, respectively (Fig. 2a). The smaller transcript predominated, and its size suggests that it originates from polyadenylation occurring close to the ATTAAA sequence present in the inserted chicken NGF fragment. This result suggests that this polyadenylation signal in the chicken NGF gene is efficiently used in vivo. The size of the larger transcript (2.2 kb) suggests that the RNA is polyadenylated at the simian virus 40 early polyadenylation signal present in the vector (Fig. 1). pERN transfections produced a single RNA transcript of 1.9 kb (Fig. 2a). The size of this transcript suggests that it includes the 771-bp rat NGF insert plus 1,150 bp from the vector preceding the SV40 early polyadenylation site. RNA prepared from cells transfected with p91023(B) without any insert (Fig. 2a, lane mock) showed no hybridization to any of the NGF probes used. Adult rooster poly(A)+ RNA, used as a hybridization control, showed the expected 1.3-kb NGF mRNA (Fig. 2a) (2). COS cell medium from pERN-transfected cells grown in the presence of [35S]methionine were immunoprecipitated with an anti-mouse NGF antibody. Analysis of the immunoprecipitate by sodium dodecyl sulfate-polyacrylamide gel
Nerve growth factor (NGF) is a 118-amino-acid protein that is essential for the development and maintenance of sympathetic neurons and a subset of sensory neurons (11, 18). Recently NGF has also been found in the central nervous system (9, 16, 20), where it appears to be essential for the function of cholinergic neurons in the basal forebrain (5, 6). The primary structure of NGF has been determined by amino acid sequencing (1). Subsequent isolation of NGF cDNA clones has allowed the structure of the NGF precursor to be deduced (15, 19) and has also shown that the mature NGF is derived by proteolytic cleavage at dibasic amino acid residues from a 305-amino-acid prepro-NGF (15). DNA probes derived from a mouse NGF cDNA clone have been used to isolate and characterize the human (19), bovine (13), rat (S. R. Whittemore, H. Persson, T. Ebendal, L. Larkfors, D. Larhammar, and A. Ericsson, in Cellular and Molecular Aspects of Neural Development and Regeneration, in press), and chicken NGF (2, 13, 22) gene. The NGF gene from all these species contains one main exon located at the 3' end of the gene encoding 243 amino acids of the prepro-NGF. Three additional short 5' exons have been identified in the mouse NGF gene (3). In this paper we report the use of a mammalian expression vector system which allows the synthesis of a recombinant NGF from either the rat or the chicken NGF gene that is similar to NGF purified from male mouse submandibular gland in molecular weight, antigenicity, and stimulation of neurite outgrowth in explanted sympathetic ganglia. A 1,046-base-pair (bp) BglI-PvuII fragment from the chicken NGF gene was converted to an EcoRI fragment by the use of EcoRI linkers followed by ligation in the unique EcoRI site of the expression vector p91023(B) (7, 8). The chicken NGF construct (pECN) contains this 3' exon of the chicken NGF gene with two translational initiation signals (ATG) separated by Qnly three nucleotides (2). In addition, a translational stop codon (TGA) and a eucaryotic polyadenyl* Corresponding author. t Present address: Department of Medical Chemistry, Laboratory of Molecular Neurobiology, The Karolinska Institute, S-104100 Stockholm, Sweden. 452
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VOL. 8, 1988 a
pECN Bgll
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pERN
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453
lation above a low endogenous NGF activity present in untransfected COS cells (Fig. 3b). Conditioned medium from transfected COS cells was preincubated with antibodies against mouse NGF to ensure that the stimulation of neurite outgrowth was due to the presence of recombinant NGF protein. The biological activity of both rat and chicken recombinant NGF was blocked by affinity-purified anti-mouse NGF antibodies (Fig. 3d and 4). The activity of purified mouse NGF was blocked at 60 to 250 ng of antibody per ml. The activity of recombinant rat NGF was blocked by 1,000 to 2,000 ng of antibody per ml, whereas chicken recombinant NGF required 2,000 ng/ml or more (Fig. 4). The presence of a mature, biologically active NGF in the medium of transfected COS cells strongly suggests that both the ATG signal and the presumptive signal sequence present in the 3' exon of the NGF gene ate functional in an in vivo system. The same ATG triplet has been shown to be used for the synthesis of a 27-kilodalton prepro-NGF protein by in vitro translation with an NGF mRNA produced in an Sp6 transcription system (3). -) b %§t.
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FIG. 1. Schematic representation of expression vector constructs containing chicken and rat-NGF sequences. (a) The 1,046-bp chicken NGF fragment (pECN) was obtained after restriction with BgIl and PvuII, and the 771-bp rat NGF fragment (pERN) was obtained after restriction with BstEII and PstI. Both fragments contain sequences from the 3' exon of the NGF gene encoding 243 amino acids of the prepro-NGF. Translational start (ATG) and stop , Region encoding the 118-amino(TGA).signals are indicated. acid mature NGF. Arrows indicate presumptive proteolytic cleavage sites. AT-TAAA refers to a polyadenylation signal present in the chicken NGF fragment. The fragments were blunt ended, EcoRi linkers were added, and the fragments were then ligated into the unique EcoRI site of p91023(B). (b) Structure of expression vector p91023(B). The features of the vector making it suitable for transient expression in COS cells have been described previously (8. 24).
electrophoresis showed the presence of a protein with the expected size for mature NGF (14 kilodaltons; Fig. 2b). A protein of the same molecular size was also immunoprecipitated from mock-transfected cells. This protein most probably represents NGF produced by the COS cells. The amount of endogenously produced COS cell NGF was approximately 20 times lower than the amount of recombinant rat NGF protein produced in pERN-transfected cells, a relation confirmed by the bioassay data described below. The biological activity of both rat and chicken recombinant NGF was measured by the ability of conditioned medium from transfected COS cells to stimulate neurite outgrowth from explanted sympathetic ganglia from 9-dayold chicken embryos. Both chicken and rat recombinant NGF proteins stimulated neurite outgrowth from the explanted sympathetic ganglia (Fig. 3a and c). From the biological activity determined in serially diluted COS conditioned medium the concentration of recombinant NGF was estimated to 5 to 40 ng/ml. The construct with the chicken NGF inserted in the opposite orientation showed no stimu-
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.
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pERN mock FIG. 2. Analysis of NGF mRNA and protein. expression in transfected COS cells. (a) Total RNA was prepared from COS cells transfected with expression vector constructs containing the rat NGF 3' exon (lane pERN) or the chicken NGF 3' exon (lane pECN) or from COS-cells transfected with the expression vector without insert (lane mock). Poly(A)+ RNA from adult rooster heart (lane heart, poly A' RNA) was included as a hybridization control. A 10-,ug portion of each sample was separated on a formaldehydecontaining agarose gel, transferred to a nitrocellulose filter, and hybridized to nick-translated NGF probes. A 900-bp Pstl-fragment from a mouse NGF cDNA clone was used for pV_RN RNA, whereas the other RNA samples were hybridized to a 910-bp Pstl-fragment from a chicken NGF clone. The filters were washed at high stringency and exposed on Kodak XAR-5 films for 3 days (lane heart. poly A' RNA) or 2 h (lanes pERN, pECN, and mock). (b) Conditioned medium was collected from COS cells transfected with the rat NGF construct (pERN) or the vector alone (mock), grown in the presence of ['5S]methionine between 48 and 60 h posttransfection. The conditioned medium was subsequently immunoprecipitated with a mouse NGF antiserum (lane anti 1-NGF) or normal rabbit serum (lane NRS). Washed immunoprecipitates were analyzed on a 10% polyacrylamide gel and fluorographed. The position of molecular size markers from 14.3 to 200 kilodaltons are shown to the left.
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FIG. 3. Biological assay of NGF activity in conditioned medium from transfected cells. Explanted sympathetic ganglia from 9-day-old
chicken embryos are shown on dark-field micrographs after incubation with conditioned medium diluted four- to eightfold. (a) Fiber outgrowth 1 day after transfection with the chicken NGF construct (pECN). (b) Lack of response (no fiber outgrowth) when using medium from cells transfected with a construct in which the chicken NGF fragment was inserted in reverse orientation for translation of NGF (mock). (c) Fiber outgrowth 2 days after transfection with the rat NGF construct (pERN). (d) Lack of fiber outgrowth after addition of 3 jig of mouse NGF antibodies per ml to conditioned medium from pECN-transfected COS cells.
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VOL. 8, 1988
04 3
2
0
4
8
16
32
64
125 250 500 1000 2000
Concentration of anti-NGF Ig (ng/mI) FIG. 4. Inhibition of recombinant NGF activity by antibodies against NGF. Recombinant rat (0) or chicken (l) NGF and purified mouse NGF (0) were incubated with increasing concentration of sheep anti-mouse NGF antibodies. The fiber outgrowth, determined as shown in Fig. 3, was scored on a scale from 0 (no fiber outgrowth) to 5 (maximal response). The values shown in the figure are mean values from two independent experiments.
The activity of chicken and rat recombinant NGF is blocked by different concentrations of the same anti-mouse NGF antibodies. These blocking profiles further support the conclusion that the NGF detected after transfection of the COS cells is the result of production of recombinant NGF and not the result of an increased expression of endogenous COS cell NGF. The efficient synthesis of a biologically active NGF in COS cells shows that this monkey cell line contains the enzymes which are necessary for correct processing of pro-NGF and that it is able to secrete NGF. The transient expression system used in this report is rapid, reproducible, and efficient and should make it possible to study the intracellular transport mechanisms involved in NGF synthesis and secretion, e.g., by immunohistochemistry. These properties also make this system suitable for testing the biological activity of NGF after performing site-directed mutagenesis of the NGF gene. Finally, the pECN and pERN constructs can be modified relatively easily to establish a stable cell line that should produce large amounts of biologically active NGF. Such cell lines can be permanently implanted into different regions of the normal and lesion-containing central nervous system (10, 17, 21) to study NGF-mediated cellular interactions and repair mechanisms in vivo. We thank Randal Kaufman, Genetics Institute, Boston, Mass., for providing the expression vector p91023(B) and James Scott for providing the mouse NGF cDNA. We also thank Dan Larhammar for advice and Katherine McIntyre for reading the manuscript. Technical assistance was given by Annika Kylberg, Stine Soderstrom, and Britt-Marie Johansson. Support was obtained from the Swedish Board of Technical Development, the Swedish Medical and Natural Science Research Councils, and the Bank of Sweden Tercentenary Foundation. LITERATURE CITED
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