Expression of a human insulin-like growth factor II cDNA in NIH-3T3 cells

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EXPRESSION

OF A HUMAN

INSULIN-LIKE GROWTH IN NIH-3T3 CELLS

FACTOR

832439

II cDNA

Daniel M. BUERGISSER, Birgit V. ROTH, Christine LUETHI, Hans Peter GERBER, Annemarie HONEGGERN and Rene E. HUMBEL Departmentof Biochemistry, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland #RarerBiotechnology Inc., Allendale Road 680, Ring of Prussia, PA 19406

Received

May 7, 1990

Recombinanthumaninsulin-like growth factor II (IGF II) wasproduced in NIH-3T3 cells transfectedwith a plasmidcontaininga constructencodingthe signalpeptideand the sequence of maturehumanIGF II. Successfullytransfectedclonessecretedcorrectly processed recombinanthumanIGF II at ratesof about 1OOngper 24 hoursper 106cells. The biological activity of the purified recombinanthumanIGF II exhibited similar potenciesasstandard humanIGF II isolatedfrom serumin radio-receptorassays aswell asin thymidine incorporation assays. 0 1990Academic Press,Inc.

The insulin-like growth factors IGF I and II are polypeptide hormonesstructurally relatedto insulin (l-4). They are producedaslarger precursorswhich, after processing,appearin serum as7.5 kDa peptidesbound to specific serumbinding proteins(5). IGF I and II display a numberof biological activities in vitro aswell asin vivo: they are ableto stimulateinsulin-lie metabolicreactionsaswell as proliferation anddifferentiation of various cells (6). In animals, acuteinjection of IGFs can result in hypoglycemia (6), and in hypophysectomizedanimals, IGF I can restore somaticgrowth (7,8). IGF I is produced under control of growth hormone (6) and mediatesmost of its actions:it is a classicalsomatomedin(9). So far, no specific function hasbeenfound for IGF II. The IGF type 1 receptor and the insulin receptor are closely relatedreceptorsboth belongingto the tyrosine kinasefamily (5). The IGF type 2 receptoris an unrelatedmolecule identified asthe cation-independentmannose-6-phosphate receptor(10, 11). Very little is known about possibleIGFeffects mediatedby this receptor (12, 13).

Abbreviations: IGF, insulin-like growth factor; rhIGF, recombinant human IGF; NIH3T3,mousefibroblast cell line; CMV, cytomegalo virus; G418, Geneticinn; TFA, trifluoracetic acid; PBS, phosphatebuffered saline;BSA, bovine serumalbumin; DMEM, Dulbecco’s modified Eagle’smedium ooO6-291XBO $1.50 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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The extensive cmssreactivities

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of IGF I, IGF II and insulin in regard to the different receptor

subtypes as well as the modulation of IGF activities by the serum binding proteins render it difficult to assign physiological responses to IGF to the activation of one particular type of receptor. One approach to overcome this difficulty is to produce mutant IGFs with enhanced specificities for one type of receptor. Other reports have aheady described the expression of IGF I mutants (14-16). We are attempting to produce IGF II mutants with enhanced specificity for the type 2 receptor. As a first step in this direction, we describe in this report the successful expression of wild-type MATERIALS

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IGF II in biologically active form. METHODS

Materials: Restriction endonucleases, M13mp18 cloning vector and PhastGel system were purchased from Pharmacia Site-directed mutagenesis kit and [sH]thymidine (5pCi/mmol) were obtained from Amersham. The expression vectors pMJ29 and pSV2neo were obtained from M. Jaye (Rorer Biotechnology Inc.). Dulbecco’s modified Eagle’s medium (DMEM) was from Animed, calf serum and geneticin (G418) from Gibco. Sep-Pak Cts cartridges and RP-300 HPLC columns were purchased from Waters Associates, n&cellulose B83 (0,2u) from Schleicher & Schuell, goat anti-mouse IgG peroxidase conjugate from Bio Rad and color development reagents from Fluka. Type 1 IGF receptor overexpressing N&I 3T3 cells (HIGR1.24) were kindly supplied by A. Ullrich (Genentech) and NIH 3T3 cells by J. Schlessinger (Rorer Biotechnology Inc.), solubilized type 2 receptor by K. von Figura (University of Giittingen). Standard hIGF I and II were preparations from human serum as previously described (4, 17). Modification of the hIGF II C-DNA: A 466 bp EcoRI-Sal1 c-DNA fragment (18) was cloned into the Ml3mpl8 cloning vector. The insert contained a 184 bp S-non-coding sequence, the 72 bp signal sequence, the 201 bp IGF II sequence and 9 bp of the E-peptide sequence. For site-directed mutagenesis, we used the improved Eckstein method (19,20) by mismatch priming. We introduced a TAG stop codon at position 457 of the insert. To clone the insert into the expression vector, we introduced at the 5’ site of the ATG translation initiation codon an XhoI restriction site and at the 3’ site of the stop codon a BamHl restriction site (Figure 1). Construction of the expression vector: The 287 bp XhoI-BamHI insert was isolated by agarose gel electrophoresis followed by electroelution and purification on a Qiagen column. The purified insert was cloned into the Xhol-Bg12 cut expression vector pMJ29 consisting of the SV40 early promotor fused at the 5’ end to the human CMV enhancer, SV40 splice and polyadenylation signal, and pML2d containing an ampicilline resistance gene (21) (Figure 1). Standard procedures for plasmid purification, ligation and transformation were used. The transformed Ecoli I-II3 101 cells were grown up in LB broth medium containing 100 pg/ml ampicilline. The hIGFII-expression vector pMJ29-IGF II was prepared by large scale procedure (22) and purified by CsCl gradient centrifugation. Cell culture and transfection: NM 3T3 cells were grown in DMEM supplemented with 10% calf serum (CS) and penicilline/ streptomycin. Recombinant plasmids were applied to NM 3T3 cells by the calcium phosphate precipitation method (23,24). 2-3 x 105 cells in 1OOmm disheswere cotransfectedwith a mixture (lOl.tg:lpg) of pMJ29-IGF II andpSV2neo (25). After overnight incubation, the cells were washedwith DMEM and 36 hoursafter transfection,the cells were split to 105cells/lOOmmdish and put underG418 (0.8mg/ml) selectivepressure.Selective mediawere changedtwice a week and after 2-3 weeks,colonies from the pMJ29-IGFzvpSV2neo cotransfectedG418-resistantcells were isolatedand cultured separatelyin selectivemedia. Isolation and purification of recombinant IGF II: The confluent cells were incubated in serum-freemediumfor 3 hours, washedand incubatedfor further 48 hours.The mediawere collectedand purified togetherwith a tracer of iuI-IGF II (26) by affiiity chromatographyas 833

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previously described (17,27). The radioactive fractions were desalted over a Cts-Sep-Pak cartridge by eluting with 60% acetonitrile/ 0.1% TFA. The eluate was concentrated to lOOl.tl and diluted with 400~1 O,l% TFA. This solution was injected on an RP-300 HPLC column and eluted in a gradient from buffer A: 0.1% TFA to buffer B: 0.1% TFA, 60% acetonitrile. Assays: The concentration of purified rhIGF II was determined by amino acid analysis and radio-immunoassays, which were done essentially as previously described (17). For the type 1 radio-receptorassay, type 1 receptor overexpressing NIH 3T3-HIGR1.24 cells were seeded at 105 cells/well into tibronectin coated 24-well dishes. After 48 hours the cells were washed with PBS/25mM HEPES pH 7.4/0.2% BSA. 150~1 1251-IGF I or II (75’000 cpm) in serum-free medium containing 0.2 % BSA was added and loOpI standard IGF II or purified rhIGF II. The cells were incubated for 5 hours at 15”, washed twice with PBS/BSA and lysed with 1ml 0.1 M NaOH. The lysate was transferred into a counting tube and measured in the y-counter. For the type 2 radio-receptorassay mannose-6phosphate/IGF II receptor purified from human liver (28) was incubated with W-IGF II and different amounts of standard IGF II or rhIGF II as described (29). [3H]thymidine incorporation into DNA of starved HIGRl.24/NIH-3T3 cells was determined as described by Riedel et al. (30). Western blot analysis: 5ml of serum-free medium from the transfected cells was desalted over Cts-Sep-Pak, concentrated to 5Ol.d and added to 25pl of reducing SDS-Laemmli buffer (31). The proteins were separated on a 7.5%-20% gradient SDS-polyacrylamide gel and then electrophoretically transferred to nitrocellulose. The blot was incubated with a 1:5000 dilution of the mouse monoclonal antibody mc43 (27) against IGF II. Reaction with peroxidasecoupled goat anti-mouse IgG and color development followed standard procedures. RESULTS Expression: The hIGF II cDNA from human brain (18) was inserted into the M13mp18 cloning vector. At position 457, a TAG stop codon was introduced to yield a construct encoding the signal peptide and the mature IGF II sequence without the C-terminal extension sequence of pro-IGF Il. Two restriction sites were constructed: an Xhol site at position 172 at the 5’ site of the ATG initiation codon and a BamHl site at position 459 at the 3’ site of the TAG stop codon (Fig. 1). The 287bp Xhol-BamHl

fragment containing the IGF II coding

sequence was inserted by sticky end ligation into the expression vector pMJ29 (21) cut with Xhol and Bgl2. Thus the resulting plasmid, pMJ29-IGF II, contained an altered cDNA fragment encoding the 24 amino acid IGF II signal peptide and the 67 amino acids of mature IGF II (Fig. 1). Restriction mapping showed the correct insertion of IGF II. After cotransfection, resistant clones were screened for IGF production. Confluent cell layers were incubated in serum-free media for 48 hours and the IGF released into the media was measured by radio-immunoassays. Nontmnsfected parental cells secreted less than lng/ml of IGF II. Conditioned media of positive clones showing significant expression (>lOOng/ml) were analyzed by SDS-PAGE and immunoblotting (Figure 2). A band corresponding in size to that of IGF II isolated from human serum (7.5 kDa) suggested that the signal peptide had been removed during the secretion process. Isolation of expressed IGF II: Serum-free medium conditioned by IGF II producing cells was purified by an immunoaffinity chromatography and reverse-phase HPLC. The major 834

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w. Constructionof thehIGF II insertandsubcloning into theexpression vector pMJ29. A: 466bpEcoRl/Sall fragmentof oneof thebraincDNA clonesof hIGFII wassubcloned into M13mpl8. ThiscDNA consists of a 184bpS-non-codingsequence, the signalpeptide (S), thefour domainsof thematurehIGFII (B,C,A,D) anda 9 bp fragmentof theE-peptide. B: To terminatetheIGF II sequence at thelengthof thepre-IGFII, thefast codonof theEpeptidewasconvertedto theTAG stopcodonby site-directed mutagenesis, anXhol anda BamHl restrictionsitewasintroducedat positions172and459to matchtheXhol/Bgl2 cloningsiteof theexpression vector. C: Structureof thehIGF II expression vector.The arrowsindicatethedirectionof transcription andtranslation.NIH-3T3cellswerecotransfected with pMJ29/IGFIIandpSV2neoas described in thetext. F&& Westernblot of differentwildtypeexpression clones.Lane1, prestained molecular weightmarker,lane2, concentrated supernatant from nontmnsfected cells;lane3,250 pg IGF II standard fromhumanserum;lanes4 and5, concentrated supematant from differentclones (~1.5and~23) expressing IGF II.

peak of the chromatograrnelutedat the samesolvent compositionashumanserumIGF II (55% buffer B = 33% acetonitrile, Fig. 3). The overall yield was 55% asmeasuredby radioimmunoassay.This isolatedrhIGF II wasnot contaminatedby IGF I or any other protein as judged by silver stainedSDS-PAGE analysisof the HPLC main and side fractions (Fig. 4), the correct aminoacid compositionand the amino-terminalsequenceof Ala-Tyr-Arg-Pro-Ser-. This last finding indicated that cleavageof the signalpeptidehad occurred at the correct site. 835

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MIN. Fig. 3. HPLC of IGF II. A: IGF II standard (2 pg). B: Recombinant IGF II of 50 ml serum-free supematant from transfected NIK3T3 cells puritied by affinity chromatography and desalted over Cts Sep-Pak. Yield: 4.6 pg (96 @ml medium).

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Fie. Silver-stained SDS-PAGE (PhastGel,, 20%) of HPLC fractions. Lanes land 6, hIGF II from serum (100 ng); lanes 2 and 3, side fractions of the main peak (1% of each side fraction); lanes 4 and 5, main fraction of rhIGF II (0.4% and 1% of the main peak); lane 7, molecular weight standard. 836

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u Radio-receptorassays of IGF II. A: Type 1 IGF receptorassay. W-IGF II displacedby hIGF II isolatedfrom serum(A, -) or rhIGF II (0,- - - ). B: Type2 IGF receptorassay. W-IGF II displacedby hIGF II isolatedfrom serum(A, - ) or rhIGF II (0, ---).

The biological activity of the purified rhIGFI1 exhibited similarpotenciesasstandardhIGF II from serumin radio-receptorassays(Figs. 5A and5B) aswell asin thymidine incorporation assays(Fig. 6). DISCUSSION Several groupshave reported the successfulexpressionof rhIGF I in various systems. Expressionof IGF I in Escherichiacoli (32-34) led to a denatured,inactive form which had to be renatured(33). A protein A-IGF I fusion protein producedin Staphylococcusaureuswas secretedand apparentlyproperly folded, but this procedurerequiredchemicalcleavageto remove the protein A region (32). Other reports describedthe expressionof rhIGF I and structural analoguesin yeast, usingthe MFal pre-pro leaderpeptideto achieve proper export andprocessing(14- 16). Expressionof rhIGF I and structural analoguesin mouseL-cell fibroblasts hasalsobeenreported (35,36), but in this systemthe useof the growth hormone signalpeptideled to a construct which wasimproperly cleaved, leaving an additionalalanine residueat the N-terminus of the IGF I sequence. Expressionof rhIGF II hasbeenreported only very recently (37,38). Hammarberget al. (37) describedthe expressionof humanIGF II asa double fusion protein in Escherichiacoli, and 837

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50

25

m

hIGFI1

[24

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ng IGFII/ml Fig. [sH]thymidineincorporationof starvedHIGR1.24/NB-i-3T.3 cells. Maximalstimulation:10%FCS.Minimalstimulation:0.5%FCS.Filledcolumns:hIGF II isolatedfrom serum.Hatchedcolumns:rhIGF II from NlH3T3.

Hummel et al. (38) have expressedIGF I and II asfusion proteinsin Escherichiacoli to be usedasantigensin the production of monoclonalan&dies. Our own attemptsto express rhIGF II in insect cells using the baculo virus systemresultedin the expressionof pre-hIGF II which was neither secretednor processed(B.R., unpublished). Proper folding and processingof recombinantIGF II were our primary aimswhich led usto choosea mammalianexpressionsystem,whereasconsiderationsof economy and efficiency were of lesserimportance. Indeedwe succeededto isolatea rhIGF II from serum-freemediumconditioned by tmnsfected mouse373 fibroblasts by a simpletwo-steppurification procedure.The total yield of the pure product was %~g/lOOml of mediumconditioned for 24 hours.No IGF I could be detectedin the mediaof the transfectedcells. The rhIGF ll wasfound to be correctly processedas determinedby amino-terminalsequenceand wasessentialypure asjudged by the aminoacid composition,the amino-terminalsequenceand the potency in comparisonto standardIGF II. ACKNOWLEDGMENTS We gratefully acknowledgethe initial studiesof Dr. PeterMaly in this work. Dr. Axe1 Ulhich kindly suppliedthe HIGRI .24 cells and Dr. Kurt von Figura a type 2 IGF receptor preparation.We alsothank Dr. JosephSchlessinger& Dr. Mike Jaye for discussionsand their help. This work wassupportedin part by the SwissNational ScienceFoundation, grant no. 3 l25739.88. REFERENCES Rinderknecht, E. & Humbel, R. E. (1976) Proc. Natl. Acad. Sci. USA 73,4379-438 1 Rinderknecht, E. & Humbel, R. E. (1978) J. Biol. Chem. 253,2769-2776 838

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Rinderknecht, E. & Humbel, R. E. (1978) FEBS Lett. 8,283-286 ~;t$el, R. E. (1984) in Hormonal Proteins and Peptides, Vol XII (C.H. Li, ed.), pp.

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Nissley, S. P. & Rechler, M. M. (1984) in Hormonal Proteins and Peptides. Vol XII (C.H. Li, ed.), pp. 127-203 Froesch, E. R., Schmid, C., Schwander, J. & Zapf, J. (1985) Ann. Rev. Physiol. 47, 443-467 Schoenle, E., Zapf, J., Humbel, R. E. & Froesch, E. R. (1982) Nature (Land.) 296, 252-253 Schoenle, E., Zapf, J., Ham-i, C., Steiner, T. & Froesch E.R. (1985) Acta Endocrinol. 108, 167-174 Daughaday, W. H., Hall, R., Salmon, W.D. Jr., Van den Brande, J. L. & Van Wyk, J.J. (1987) J. Clin. Endocrinol. Metab. 65, 1075-1076 Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A. & Rutter, W. J., 1987, Nature (Lond.) 329, 301-307 Mac Donald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P. & Ullrich, A. (1988) Science 239,1134- 1137 Kojima, I., Nishimoto, I., Iiri, T., Ogata, E. & Rosenfeld, R., (1988) Biochem. Biophys. Res. Commun. 154,9-19 Tally, M., Enberg, G., Li, C.H. & Hall, K. (1987) B&hem. Biophys. Res. Commun. 147, 1206-1212 Bayne, M. L., Applebaum, J., Chicchi, G. G., Hayes, N. S., Green, B. G. & Cascieri, M. A. (1988) J. Biol. Chem. 263,6233-6239 Cascieri, M. A., Chicchi, G. G., Applebaum, J., Hayes, N. S., Green, B. G. & Bayne, M. L. (1988) Biochemistry 27,3229-3233 Cascieri, M. A. Hayes, N. S. & Bayne, M. L. (1989) J. Biol. Chem. 264,2199-2212 Zumstein, P. P. & Humbel, R. E. (1985) Methods in Enzymology 109,782-798 Irminger, J. C., Rosen, K. M., Humbel, R. E. & Villa-Komaroff, L. (1987) Proc. Natl. Acad. Sci. USA 84,6330-6334 Taylor, J. W., Ott, J. & Eckstein, F. (1985) Nucleic Acids Res. 13, 8764-8785 Nakamaye, K. & Eckstein, F. (1986) Nucleic Acids Res. 14,9679-9698 Jaye, M., Lyall, R. M., Schlessinger, J. & Sarver, M. (1988) EMBO J. 7, 963-969 Davis, L. G., Dibner, M. D., Battey, J. F. (1986), in Basic Methods in Molecular Biology, pp. 93-98, Elsevier Science Publishing Co. Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G. & Chasin, L. (1979) Proc. Natl. Acad. Sci. USA 76, 1373-1376 Sarver, N., Byrne, J. C. & Howley, P. M. (1982) Proc. Natl. Acad. Sci. USA 79, 7147-7151 Southern, D. J. & Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341 Zapf, I., Walter, H. & Froesch, E. R. (1981) J. Clin. Invest. 68, 1321-1330 Laeubli, U. K., Baier, W., Binz, H., Celio, M.R. & Humbel, R.E. (1982) FEBS Letters 149, 109-112 Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Hasilik, A. & von Figura, K., (1984) I. Cell Biol. 98,2047-2054. Maly, P. & Liithi, C. (1986) Biochem. Biophys. Res. Commun. 138, 1257-1262 Riedel, H., Massoglia, S., Schlessinger, J. & Ulhich, A. (1988) Proc. Natl. Acad. Sci. USA 85,1477-1481 Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 Buell, G. Schultz, M. F., Selzer, G., Chollet, A., Mowa, M. R., Semon, D., Escanez, S. & Kawashima, E. (1985) Nucleic Acids Res. 13,1923-1938 Peters, M. A., Lau, W. P., Snitman, D. L., Van Wyk, J. J., Underwood, L. E., Russell, W. E. & Svoboda, M. E. (1985) Gene (Amst.) 35,83-89 Nilson, B., Holmgren, E., Josephson, S., Gatenbeck, S., Philipson, L. & Uhlen, M. (1985) Nucleic Acids Res. 13, 1151-l 162 Bayne, M. L., Cascieri, M. A., Kelder, B., Applebaum, J., Chicchi, G. C., Shapiro, J., Pasleau, F. & Kopchick, J. J. (1987) Proc. Natl. Acad. Sci. USA 84, 2638-2642 Cascieri, M. A., Hayes, N. S., Kelder, B., Kopchick, J. J., Chicchi, G. C., Slater, E. E. & Bayne, M. L. (1988) Endocrinology 122, 1314-1320 Hammarberg, B., Nygren, P., Holmgren, E., Elmblad, A., Tally, M., Hellman, U., Moks, T & Uhlen, M. (1989) Proc. Natl. Acad. Sci. USA 86,4367-4371. Hummel, M., Herbst, H. & Stein, H. (1989) Eur. J. B&hem. 180, 555-561

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