J. Biochem. 107, 486-492 (1990)

Structural Characterization of Natural and Recombinant Human Granulocyte Colony-Stimulating Factors1 Naoki Kubota,2 Tetsuro Orita, Kunihiro Hattori, Masayoshi Oh-eda, Norimichi Ochi, and Tatsumi Yamazaki Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Komakado 1-135, Gotemba, Shizuoka 412 Received for publication, September 12, 1989

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which stimulates predominantly neutrophilic granulocyte colony formation in mammals. Natural human G-CSF (hG-CSF) and recombinant hG-CSF produced in Chinese hamster ovary (CHO) cells transfected with the cDNA clone for hG-CSF have been purified to apparent homogeneity for structural and biological comparison. The amino acid sequence of recombinant hG-CSF, composed of 174 amino acid residues, was identical with that of natural hG-CSF and also with the sequence predicted from the cDNA. Both forms of hG-CSF have a free Cys-17 and two intramolecular disulfide linkages, between Cys-36 and Cys-42, and between Cys-64 and Cys-74. The O-glycosylation occurred at Thr-133 in both hG-CSFs. Similar CD spectra were obtained for both hG-CSFs. Additionally, both forms showed almost the same biological activities determined by in vitro colony-forming assay and in vivo assay. It is thus concluded that the recombinant hG-CSF is indistinguishable from its natural counterpart and that the former is valuable for more detailed characterization and clinical use.

The colony-stimulating factors (CSF) are hormone-like glycoproteins that regulate the proliferation and differentiation of hematopoietic progenitor cells. Four CSFs are known: granulocyte-macrophage CSF {1, 2), granulocyte CSF (G-CSF) (3), macrophage CSF (4, 5), and multi-CSF (or interleukin 3) (6, 7). G-CSF acts specifically on the granulocyte lineage and has recently been shown to enhance some functions of the mature granulocyte (8, 9). Many growth factors and lymphokines have been produced by recombinant DNA technology, but few structural comparisons of the recombinant protein with the natural counterpart have been reported {10-12). Unexpected products different from the deduced amino acid sequence from the respective cDNA sequence have sometimes been obtained in the expression of the recombinant proteins (13, 14). In the cases where proteins are produced in prokaryotic cells such as E. coli, proper post-translational modifications, i.e., folding and glycosylation of proteins, are not expected. The resulting improper molecular forms may lead to biological dysfunction, especially antigenicity, when used therapeutically (15-17). We have previously reported the cloning of genomic and complementary DNA for hG-CSF and their expression in mammalian cells (28, 29). Recombinant hG-CSF produced in mammalian cells is believed to undergo the proper post-translational modifications. However, CHO cells are 1 Portions of this paper (Tables IS-IVS and Fig. 1S-6S) are presented in the miniprint at the end of the paper. 2 To whom correspondence should be addressed. Abbreviations: G-CSF, granulocyte colony-stimulating factor; hGCSF, human granulocyte colony-stimulating factor; CHO, Chinese hamster ovary; PTH, phenylthiohydantoin; PTC, phenylthiocarbamoyl; RCM, reduced and carboxymethylated; TFA, trifluoroacetic acid; CPA, cyclophosphamide; NeuAc, N-acetylneuraminic acid; GalNAc, JV-acetylgalactosamine; GaLNAcol, N-acetylgalactosaminitol.

different from the human cells producing the factor naturally. It is important to compare not only the biological but also the structural properties of recombinant hG-CSF with its natural counterpart. Oh-eda et al. (20) have already reported that the carbohydrate structures of the hG-CSFs are similar. In this paper we describe the structural characterization of recombinant hG-CSF produced in CHO cells in comparison with its natural counterpart. The biological activities of both hG-CSFs will also be discussed. MATERIALS AND METHODS Purification of Human G-CSF—Natural and recombinant hG-CSFs were purified from the conditioned media of the human squamous carcinoma cell line CHU-2 and CHO cells transfected with the cDNA clone for hG-CSF as described previously (21). Amino Acid Analysis—Samples were hydrolyzed in 6 N HC1 containing 0.2% phenol for 24 h at 110'C in evacuated sealed glass tubes. The resulting amino acids were phenylisothiocyanated to phenylthiocarbamoyl derivatives. The derivatives were separately quantified in a PICO-TAG amino acid analysis system (Waters Associates) (22). A'-Terminal Sequence Analysis—Samples were subjected to automated Edman degradation in an Applied Biosystems (ABI) Protein Sequencer 470A. The resulting phenylthiohydantoin (PTH) derivatives were identified and quantified by narrow bore reverse phase HPLC in an ABI 120A PTH-Analyzer using a mixture of standard PTH amino acids (Pierce Chemical) (23). C-Terminal Sequence Analysis—The lyophilized samples of 0.5 mg of recombinant hG-CSF or natural hG-CSF were dissolved in 1 ml of 0.05 M sodium phosphate buffer (pH 6.5) containing 2% SDS, heated for 3 min at 100'C,

486 Downloaded from https://academic.oup.com/jb/article-abstract/107/3/486/806895 by University of Chicago user on 09 April 2018

J. Biochem.

487

Structures of Granulocyte Colony- Stimulating Factors diluted 4-fold with 0.05 M sodium phosphate buffer (pH 6.5), and digested with carboxypeptidase Y (Oriental Yeast) at an enzyme/substrate ratio of 1 : 20 (w/w) at 25'C. The samples were withdrawn at fixed time intervals and quantified by amino acid analysis as described above. Reduction and S-Carboxymethy lotion—Recombinant hG-CSF (2 mg) was reduced and caboxymethylated with iodoacetic acid by the method of Crestfield et al. (24). The reduced and carboxymethylated (RCM)-derivative was purified by reverse phase HPLC and lyophilized. RCMnatural hG-CSF was prepared in the same manner. Enzymatic Digestion—RCM-recombinant hG-CSF (1 mg) was digested with Staphylococcus aureus V8 protease (Boehringer) in 0.05 M ammonium bicarbonate (pH 7.8) for 18 h at 37"C at an enzyme/substrate ratio of 1 : 50 (w/ w). The RCM-protein (1 mg) was' also digested with ff-chymotrypsin (Sigma) in the same buffer for 3 h at 37'C at an enzyme/substrate ratio of 1 : 100 (w/w). Peptides were separated by reverse phase HPLC using Capcellpak C18 (4.6x250 mm, Shiseido). The column was equilibrated with 5% acetonitrile in 0.1% trifluoroacetic acid (TFA), and the peptides were eluted at a flow rate of 0.6 ml/min with a linear gradient of acetonitrile from 5 to 80% in 0.1% TFA over 120 min. Peptides were detected by their absorbance at 220 and 280 nm. Peptides derived from the protease digests of the RCM-natural hG-CSF (1 mg) were also prepared in the same manner. Disulfide Bond Analysis—Free sulfhydryl group in the recombinant hG-CSF was determined by the method of Ellman et al. (25). The carboxymethylated recombinant hG-CSF (1 mg) without reduction was digested with S. aureus V8 protease, and the resulting peptides were separated by reverse phase HPLC as described previously. The peptide containing carboxymethylated cysteine was identified by amino acid analysis. One milligram of the intact recombinant hG-CSF was digested with S. aureus V8 protease or cr-chymotrypsin, and the digest was separated by reverse phase HPLC to obtain cystine-containing peptides. One cystine-containing peptide (E3) was further digested with a-chymotrypsin in 0.05 M ammonium bicarbonate buffer (pH 7.8) for 3 h at 37*C. The other cystinecontaining peptide (C23) was further digested with thermolysin in 0.1 M iV-ethylmorpholine buffer (pH 8.0) for 3 h at 37'C. The cystine-containing peptides were also obtained from 1 mg of natural hG-CSF in the same manner. Determination of the Glycosylation Site—The glycosylated peptide was identified by the determination of sialic acid and iV-acetylgalactosamine (GalNAc) in peptides obtained 10 3 xM,

92.5 66.2 _ 45.0 ~ 31.0

-

21.5

-

14.4

A

B

C

Fig. 1. SDS-PAGE of hG-CSF. Proteins about 1 n% were electrophoresed on 13.5% gel and stained with Coomassie Brilliant Blue R-250. Lane A, molecular weight markers; lane B, recombinant hG-CSF; lane C, natural hG-CSF.

Vol. 107, No. 3, 1990 Downloaded from https://academic.oup.com/jb/article-abstract/107/3/486/806895 by University of Chicago user on 09 April 2018

by the digestion of recombinant hG-CSF with S. aureus V8 protease. Sialic acid and GalNAc were determined by the periodate-resorcinol method (26) and by the amino acid analysis, respectively. Glycosylated peptide of about 5 nmol was dissolved in 0.05 ml of 0.2 M Tris-maleate buffer (pH 6.0) and was successively digested with 50 milliunits of neuraminidase (Seikagaku Kogyo) for 1.5 h and 5 milliunits of O-Glycanase (Endo-ff-iV-acetylgalactosarninidase, Genzyme) for 24 h at 37*C. Deglycosylated peptide was isolated by reverse phase HPLC as described. The Oglycosylation site was determined by sequencing of the glycosylated peptide before and after removal of the carbohydrate chain. CD Spectra—CD spectra of recombinant and natural hG-CSF were taken in a JASCO J-500A spectropolarimeter at room temperature. The protein concentration of each factor was 0.2 mg/ml in distilled water (pH 7.0). Cyclophosphamide-Mouse Assay—Male c57BL/6N mice (7-9 wk old) were injected with cyclophosphamide (CPA) intraperitoneally at 100 mg/kg of body weight on day 0. The mice were injected with 0.2 ml of hG-CSF solution subcutaneously on day 1 to day 4. The mice were bled retro-orbitally, and total leukocytes were counted 6 h after the last injection in a Microcellcounter (CC-180A, Toa Medical Electronics). Colony-Forming Assay—The colony-forming activity of the hG-CSFs was determined as described previously (21). RESULTS Purification of Natural and Recombinant hG-CSF— Natural and recombinant hG-CSFs were purified to homogeneity. The purified proteins gave a single band on SDS-PAGE as shown in Fig. 1. The amino acid compositions of the purified factors are shown in Table I, and no essential difference was observed. Primary Structure of hG-CSFs—The N- and C-terminal sequences of recombinant and natural hG-CSFs were TABLE I. Amino acid composition of recombinant and natural hG-CSFs. Each amino acid was determined as PTC-amino acid. The values with asterisks were used for molar ratio calculation, as references deduced from the cDNA sequence. The number of Cys and Trp residues were not determined. Amino acid

Asx (D, N) Glx (E, Q) Ser(S) Gly (G) His (H) Arg(R) Thr (T) Ala (A) Pro(P) Tyr(Y) Val (V) Met (M) Cys(Q He (I) Leu(L) Phe (F) Trp(W) Lys (K) Total

Predicted from cDNA 4

26 14

14 5

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13 i 1 I 5 4 33 6

2 4 174

Determined Recombinant Natural 4.1 4.2 27.0 26.8 13.2 13.4 14.9 14.5 5.2 4.9 4.9 4.8 6.7 6.5 19.0* 19.0' 13.2 12.8 2.8 2.9 6.5 6.9 3.0 2.7 — — 3.5 3.7 32.4 32.1 6.4 5.9 — — 4.5 5.1

N. Kubota et al.

488 1 5 10 15 20 Tlr-Pro-L«i-Cl7-Pro-il»-5er-S«r-Ui-Pro-fili-S«r-Pie-L«i-i«i-Lj«-tji-Ui-—

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Fig. 5. Dose-response curves for the colony-stimulating activities of recombinant and natural hG-CSF. Marrow cells (1 x 10») from C57BL/6N mice were cultured with serial twofold dilutions of recombinant (o) or natural ( • ) hG-CSF. Duplicate cultures were scored on day 5 of the culture.

group was detected by the titration of intact hG-CSF with 5,5'-dithiobis(2-nitrobenzoic acid), the other four cysteine residues were considered to form two pairs of intramolecular disulfide bonds. One mole of carboxymethylcysteine was detected only in the peptide containing Cys-17 by the amino acid analysis of the peptides obtained by S. aureus V8 protease digestion of the non-reduced and RCM hGCSFs. These data indicate that only Cys-17 is free. The cystine-containing peptides E3 and C23 were obtained by the digestion of intact factors with S. aureus V8 protease (Fig. 3S) and a-chymotrypsin (Fig. 4S), respectively. The E3 and C23 peptides were further digested with achymotrypsin and thermolysin, respectively. The resulting digests were separated by reverse phase HPLC as shown in Fig. 5S, and the two cystine-containing peptides were sequenced as below. 36

K-L-C-A-T-Y

64 S- S - C - P - S - Q - A

K-L-C-H-P-E-E 42

L- A - G - C 74

1

h

Amino acid sequences of the E3 and C23 peptides were concluded to be as follows. 36 42 K-L-CA-T-Y-K-L-CH-PE-E 64 74 S-S-C-P-S-Q-A-L-Q-L-A-GC-L-S-Q-L-H-S-G-L-F

The results of the sequence analysis are shown in Table

ins. Based on these data, it is concluded that the two disulfide bonds are formed between Cys-36 and Cys-42, and between Cys-64 and Cys-74. Glycosylation Site—The O-glycosylation site of recombinant hG-CSF has not been determined yet although Oh-eda et al. have reported the carbohydrate structure of the hG-CSF (20). To obtain the glycosylated peptide, the peptides derived from S. aureus V8 protease digestion of intact recombinant hG-CSF were quantified for sialic acid Vol. 107, No. 3, 1990 Downloaded from https://academic.oup.com/jb/article-abstract/107/3/486/806895 by University of Chicago user on 09 April 2018

I Dole of hG-CSF ( ng/mouie/diy)

10

Fig. 6. Hie comparative effects of recombinant and natural hG-CSF on peripheral blood leukocyte levels in CPA-treated mice. The mice were injected daily for 4 days with various doses of recombinant ( • ) or natural (o) hG-CSF 1 day after the CPA injection. Blood samples were obtained 6 h after the last dose of each form of hG-CSF.

and GalNAc. The sugars were detected only in E8 peptide (residues 124-162, Fig. 2). The 10th amino acid residue from the N-terminus of the E8 peptide was unidentified, but Thr was detected at this position after removal of the sugar chain by the successive digestion with neuraminidase and O-Glycanase (Fig. 6S and Table IVS). The same results were also obtained for natural hG-CSF. The O-glycosylation site of both hG-CSFs is thus concluded to be Thr-133. CD Spectra—The CD spectrum of the recombinant hG-CSF was identical to that of natural hG-CSF, as shown in Fig. 4. The change of the ellipticity at 222 nm by thermal denaturation was almost the same between recombinant and natural hG-CSF (data not shown). These results suggest that the conformation of the recombinant hG-CSF is similar to that of natural hG-CSF. Biological Activities—Natural and recombinant hGCSFs had similar dose-response curves in the colony-forming assay, as shown in Fig. 5. The maximal colony numbers and the slopes were essentially the same in both hG-CSFs. Figure 6 shows the in vivo activity of the hG-CSFs in the CPA-mouse assay. Dose-dependent increase of peripheral leukocytes was observed with both hG-CSFs, indicating that the two factors have almost the same specific activity. DISCUSSION The expression of recombinant G-CSF produced in E. coli and mammalian cells has been reported by several groups (27-29). However, structural characterization of the protein has been focused on only G-CSF derived from E. coli (30-31), and there have been no experiments comparing natural and recombinant G-CSF. In this paper we have described the structural comparison of natural hG-CSF produced in CHU-2 with recombinant hG-CSF produced in CHO cells. We determined the primary and secondary structures of both forms of hG-CSF. In hG-CSF all sites sensitive to trypsin were localized mainly in the N- and C-terminal regions; thus we have used S. aureus V8 protease for the fragmentation of the protein. Digestion with this protease yielded peptides of appropriate size for amino acid se-

N. Kubota et al.

490 quence determination. The elution profile of the S. aureus V8 protease digest of the recombinant hG-CSP was in good agreement with that of the natural hG-CSF on reverse phase HPLC, and had high reproducibility. Two disulfide linkages were determined by analyzing the peptides obtained by digestion of the intact protein with S. aureus V8 protease or o'-chymotrypsin. It was reported that the disulfide linkages are unstable at alkaline pH and that the free sulfhydryl group causes disulfide exchange reaction (32, 33). In the present conditions where S. aureus V8 protease digestion was performed at pH 7.8, no disulfide exchange reaction of hG-CSF was observed. Furthermore, we obtained the same results by pepsin digestion at pH 2.0 (data not shown). We previously reported that hG-CSF contained the O-linked carbohydrate chain NeuAca-2-3Galy31-3(±NeuAca2-6)GalNAcol (20). In this paper the glycosylation site was determined by sequencing the peptide containing carbohydrate chain before and after enzymatic removal of the sugar chain from the peptide. The possible iV-glycosylation site is known to be asparagine at the consensus sequence, Asn-X-Ser/Thr. Although no consensus sequence is known for the 0-glycosylation, it has been proposed that the O-linked carbohydrate chain is bound to a serine or threonine residue near a proline residue (34). The O- glycosylation site of the recombinant and natural hG-CSFs was determined as Thr-133 near Pro-132. Kagawa- et al. have reported that human interferon p produced in three kinds of mammalian cells had different specific activities and carbohydrate structures (35), and they concluded that CHO cells produced a protein resembling the natural one. The CHO cell expression system is thought to be valuable for the expression of human glycoproteins. There are no differences between the recombinant and natural hG-CSFs in the entire amino acid sequence, the positions of the disulfide bonds, or the O-glycosylation site. Comparison of the CD spectra also suggests no difference between the two forms. Additionally, the two forms showed the same biological activities in the in vitro colony-forming assay and in vivo CPA-mouse assay, supporting the structural comparison data. It is thus concluded that the recombinant hG-CSF is indistinguishable from its natural counterpart.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. We thank Drs. S. Hata, M. Ono, R. Kaifu, and T. Tanaka (Chugai Pharmaceutical Co., Ltd.) for helpful discussions and encouragement.

29.

REFERENCES

30.

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Wilson, K.J., Boosman, A., Stanley, E.R., Ralph, P., & Mark, D.F. (1985) Science 230, 291-296 Ihle, J.N., Keller, J., Henderson, L., Klein, F., & Palaszynski, E. (1982) J. Immunol. 129, 2431-2436 Fung, M.C., Hapel, A.J., Ymer, S., Cohen, D.R., Johnson, R.M., Campbell, H.D., & Young, I.G. (1984) Nature 307, 233-237 Kitagawa, S., Yuo, A., Souza, L.M., Saito, M., Miura, Y., & Takaku, F. (1987) Biochan. Biophys. Res. Commun. 144, 11431146 Yuo, A., Kitagawa, S., Okabe, T., Urabe, A., Komatsu, Y., Itoh, S., & Takaku, F. (1987) Blood 70, 404-411 Utsumi, J., Yamazaki, S., Hosoi, K., Shimizu, H., Kawaguchi, K., & Inagaki, F. (1986) J. Biochan. 99, 1533-1535 Langley, K.E., Lai, P.H., Wypych, J., Everett, R.R., Berg, T.F., Krabill, L.F., Davis, J.M., & Souza, L.M. (1987) Eur. J. Biochan. 163, 323-330 Becker, G.W. & Hsiung, H.M. (1986) FEBS Lett. 204, 145-150 Yamazaki, S., Shimazu, T., Kimura, S., & Shimizu, H. (1986) J. Interferon Res. 6, 331-336 Recney, M.A., Scoble, H.A., & Kim, Y. (1987) J. BioL Chan. 262, 17156-17163 Morehead, H., Johnston, P.D., & Wetzel, R. (1984) Biochemistry 23, 2500-2507 Freisen, H.J., Stein, S., Evinger, M., Familletti, P.C., Moschera, J., Meienhofer, J., Shively, J., & Pestka, S. (1981) Arch. Biochan. Biophys. 206, 432-450 Colby, C.B., Inoue, M., Thonpson, M., & Tan, Y.H. (1984) J. Immunol. 133, 3091-3095 Nagata, S., Tsuchiya, M., Asano, S., Kaziro, Y., Yamazaki, T., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., & Ono, M. (1986) Nature 319, 415-417 Nagata, S., Tsuchiya, M., Asano, S., Yamamoto, 0., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., & Yamazaki, T. (1986) EMBO J. 5, 575-581 Oheda, M., Hase, S., Ono, M., & Ikenaka, T. (1988) J. Biochan. 103, 544-546 Nomura, H., Imazeki, I., Oheda, M., Kubota, N., Tamura, M., Ono, M., Ueyama, Y., & Asano, S. (1986) EMBO J. 5, 871-876 Heinrikson, R.L. & Meredith, S.C. (1984) Anal. Biochan. 136, 65-74 Hewick, R.M., Hunkapiller, M.W., Hood, L.E., & Dreyer, W.J. (1981) J. Biol. Chan. 256, 7990-7997 Crestfield, A.M., Moore, S., & Stein, W.H. (1963) J. Biol. Chan. 238, 622-627 Ellman, G.L. (1959) Arch. Biochan. Biophys. 82, 70-77 Jourdian, G.W., Dean, L., & Roseman, S. (1971) J. BioL Chan. 246, 430-435 Souza, L.M., Boone, T.C., Gabrilove, J., Lai, P.H., Zsebo, K.M., Murdock, D.C., Chazin, V.R., Bruszewski, J., Lu, H., Chen, K.K., Barendt, J., Platzer, E., Moore, M.A.S., Mertelsmann, R., & Welte, K. (1986) Science 232, 61-65 Tsuchiya, M., Nomura, H., Asano, S., Kaziro, Y., & Nagata, S. (1987) EMBO J. 6, 611-616 Devlin, P.E., Drummond, R.J., Toy, P., Mark, D.F., Watt, K.W.K., & Devlin, J.J. (1988) Gene 65, 13-22 Wingfield, P., Benedict, R., Turcatti, G., Allet, B., Mermod, J.J., Delamarter, J., Simona, M.G., & Rose, K. (1988) Biochan. J. 256, 213-218 Lu, H.S., Boone, T.C., Souza, L.M., & Lai, P.H. (1989) Arch. Biochan. Biophys. 268, 81-92 Ryle, A.P. & Sanger, F. (1955) Biochan. J. 60, 535-540 Spackman, D.H., Stein, W.H., & Moore, S. (1960) J. BioL Chan. 236, 648-669 Young, J.D., Tsuchiya, D., Sandlin, D.E., & Holroyde, M.J. (1979) Biochemistry 18, 4444-4448 Kagawa, Y., Takasaki, S., Utsumi, J., Hosoi, K., Shimizu, H., Kochibe, N., & Kobata, A. (1988) J. Biol. Chan. 263, 1750817515

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491

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14

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2

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(its)

HOI) (1911) (1174) 12741)

(ton) (2(52)

La (mi) La (im: CU IKK) Cll (1741) fr» (117!) Fro (14911

nr

(142])

CU ( US) Cll (Mill

1] ] 7 1 4 11 1 1 4 174

0.1 i • l . l l l ) 0.1(1) i . i •I.Ill) •l.llll 1.1(1) •4 014) •I OKI • .nil H (!) K ID 0 1 2.111) 4.mi • mi 0.0 1.1 .mi

1.0 •1.0(2)

(in)

(141) (1(9) (IS) (171) (101) tn Tir 1 41) CU ( 82)

a*

a

II

Fnctliu El

on) (IB)

I I

14 i S 7

Table.IIS Aalno acid composition analyses of peptldes obtained by digestion of the RCM-natural hG-CSF with S. aureus V8 protease.

icU

— Cii

IIS)) (IN) (1211

EJ*ni ittilnl tj Uititlu if a H)tHi >ltt inruliluit

Eick IIIM K N ••! Hltnliri* it PTC-alH icU u4 Is rtrrtiutH Ii PHI. Tiliet vita uterllki >trt ml fir Hllr n l l i Cllcllitln, u rtftructl ithcii tm lit d l l i m u c t 10: lit MUtiritl.

liln

1) iit.nl K-CV

liln ulifctntni(ml) Cjtlt B a*

Tltll 4 2f 1 14 14 1 5 7 11 11 1 7 1

ISO

240

300

380

Ttm (n*l> Fir. IS C-termlnal sequence analysis of recoablnant hG-CSF SDS-denatured reconblnant hG-CSF was digested with carboxypeptldase Y at an enzyme/substrate ratio of 1 : 20(w/w) at pH 6.5 and 25°C The samples wero withdrawn at the tines Indicated ln the flrure and released amlno acids were quantified by the Plco-Tai method

0.94

4

11 I I 4 174

0

Eick u l u ul. *u .itenlid n nt-ulii icH ul Ii rifttiatti Ii p» Tilrti vltk uterllki vert nM fir ulir ntlt cikilitln, u nferacu Midi tm ii* cHA iifiuciIQ: lit witlflH. Table H I S Aalno acid sequence analysis of peptldes obtained by dlrestlon of E3 peptlde with ci-chymotrypsln. 1) rtnikluit ttf-OF Cjcll 1 2 1 4

1 ( 7

i l l u icH (i LTI (UK) La (I9KI 1 CTI ( i l l (lilt)

nr ( nt) TTT ( sat

I) Mtinl K-QF

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(in)

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Aalno acid sequence analysis of peptldes obtained by digestion of C23 peptlde with thennolysln. 0 -tttrilm t K-OF Cjcll

1

111 u

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nm)

Vol. 107, No. 3, 1990 Downloaded from https://academic.oup.com/jb/article-abstract/107/3/486/806895 by University of Chicago user on 09 April 2018

O 40 80 80 Retention Tim* ( mtn )

100

Fll. 2S Separation of t^-chynotryptlc peptldes of the RCMrecomblnant and RCM-natural hG-CSF. The HCM-recomblnant hG-CSF or ROt-natural hC-CSF was digested with a -chynotrypsln at an enzyme/substrate ratio of 1 : 100 (w/w) at pH 7.8 and 37*c for 3 h. Peptldes were separated by reverse pha«e HPLC. The column was equilibrated with SX acetonltrlle In O.l\ TFA, and the peptldes were eluted a flow rate 0.6 El/uln with a linear trad lent of acetonltrlle fron 5X to BOA ln O.1X TFA over 120 mln. A. RCM-reconblnant hG-CSF: B, RCM-natural hG-CSF

S.

492

N. Kubota et al.

044 044

• 10

0.31

I

B4

-40

044

042

...--"iio

40

100

60

Retention TInw ( mJn )

1 • l l . LJ 20

40

80

Fir. 4S BO

100

Retention TInw ( mtn )

Fit. 3S Separation of S. aureua V8 protease peptldea of Intact recoablnant and natural hG-CSFs. Intact reconbinant or natural hC-CSF was dlffeated with S. aureus V8 protease at an enzyme/substrate ratio of 1 : 50 (w/w) at pH 7.8 and 37 °C for 18 h. Peptldea were separated by reverse phase HPLC. The coluan was equilibrated with 5* acetonltrlle In 0 . H TFA. and the peptldes were eluted a flow rate 0.6 ad/iln with a linear tradlent of acetonltrlle frcw 6X to SOX In 0.1X TFA over 120 mln. A, reccmblnant hG-CSF: B. natural hG-CSF.

Separation of tf-chymotrypsin peptldea of Intact recomblnant and natural hG-CSFa. Intact recoablnant or natural hG-CSF was dlrested with a-chymotrypsln at an enzyae/aubatrate r a t i o of 1 • 100(w/w) at pH 7.8 and 37 "c for 3 h. Peptldea were separated by raverae phase HPLC. The column waa equilibrated with 5X acetonltrlle In 0.1* TFA and the peptldea were eluted a flow rate 0.6 Bl/»ln with a linear gradient of acetonltrllo fron 6X to SOX In 0 IX TFA over 120 Bin. A, reconblnant hG-CSF; B, natural hG-CSF.

20 40 eo RatMitlon Tkna ( Bin ) Fig

5S Separation of peptldes obtained by digestion of E3 peptlde wlthoz-chyaotrypsln and separation of peptldea obtained by dltestlon of C23 peptlde with theraolysln. E3 peptlde of recomblnant or natural hG-CSF was digested with tf-chymotrypsln at enzrne/aubstrate ratio of 1 : 100 (w/w)at pH 7.8 and 37*C for 3 h. The C23 peptlde of recoablnant or natural hG-CSF waa digested with thernolysln at an enzyae/substrate ratio of 1 : 100(w/w) at pH 8.0 and 3T°C for 3 h. Peptldei were separated by reverse phase HPLC. The column was equilibrated with SX acetonltrlle In 0.1X TFA and the peptldes were eluted at a flow rate of 0.8 Bl/mln with a linear gradient of acetonltrlle from SX to 80X in 0.1X TFA over 120 Bin. A.B: Elutlon pattern of peptldes obtained by digestion of E3 peptlde. A.recomblnant.and B.natural hG-CSF. C D : Elutlon pattern of peptldes obtained by digestion of C23 peptlde. C.recomblnant.and D.natural hG-CSF. Peak 2 was a cystlno-contalnlng peptlde.

to

40 so R»t«ntron Ttmt ( mtn )

to

Fig. 6S Separation of peptldes obtained by digestion of E8 peptlde with neuraalnldase and 0-Glycanase. E8 peptlde was successively digested with nenraalnidase for 1.5 h and 0-Glycanase for 24 h at 37*C. Deslycosylated peptlde E8 was Isolated by reverse phase HPLC as described In Fit. 6S. A, recoablnant hG-CSF; B. natural hC-CSF.

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