Molecular Characterization of Recombinant Human Acidic Fibroblast Growth Factor Produced in E. co//: Comparative Studies with Human Basic Fibroblast Growth Factor

Tatsuya Watanabe, Masaharu Seno, Reiko Sasada, and Koichi Igarashi Biotechnology Research Laboratories Research and Development Division Takeda Chemical Industries, Ltd. Yodogawa-ku, Osaka 532, Japan

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 Mg/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin. (Molecular Endocrinology 4: 869-879, 1990)

exhibit pleiotropic biological activities in vitro for a variety of cell types derived from mesoderm and neuroectoderm (1, 2). They also induce angiogenesis in vivo (reviewed in Ref. 3). Interaction with the glycosaminoglycan heparin is one of the most prominent characteristics of FGFs. The affinity of FGFs for heparin is unusually high, so that a concentration of NaCI between 1-1.5 M is required to elute aFGF or bFGF from a heparin-Sepharose affinity column (2, 4). Heparin-binding protects FGFs in vitro from degradation by certain proteolytic enzymes (5-7) and from inactivation by heat and acid (5-8). Heparin and related glycosaminoglycans are known to appear on the extracellular matrix, and they have been proved to act physiologically as a reserver for bioactive FGFs (9). Interestingly, heparin affects the biological activities of aFGF and bFGF differently. The biological activity of aFGF was reported to be potentiated by heparin (10), although heparin has been reported to inhibit the activity of bFGF (11). Although the mechanism of this phenomenon is not yet established, it has been speculated that a heparindependent conformational change in aFGF may be involved (12). We previously described the bacterial production and characterization of human (h) bFGF (12), and in this report we have purified and characterized haFGF produced in E. coli in order to facilitate the comparison of FGFs.

INTRODUCTION

RESULTS

Acidic and basic fibroblast growth factor (aFGF and bFGF) are structurally related heparin-binding polypeptides with a similar range of biological activity. They

Purification of haFGF We used the T7 phage RNA polymerase-promoter system for bacterial expression of haFGF cDNA. Synthetic haFGF cDNA was properly integrated into plasmid pET3c (13) downstream from the T7 promoter to con-

0888-8809/90/0869-0879$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

869

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Vol 4 No. 6

MOL ENDO-1990 870

Ndel

BamHI

Ncol

Ndel

BamHI

large fragment

Ncol linker

— Ncol — large fragment — BamHI

BspMI Hindi 11

blunt Ncol

BamHI

BamHI

BspMI large fragment BamHI

BamHI

Fig. 1. Construction of Expression Plasmid Plasmid pET3c was modified by insertion of an A/col translation start adaptor (5'-CCATGG-3') at the preexisting Nde\ site to generate a convenient cloning site. The resulting plasmid vector (pET3c,A/col) was cleaved by A/col, end-filled with DNA polymeraseI large fragment, and finally digested with BamH\ to be ready for the assembly of a haFGF open reading frame. To prepare haFGF synthetic cDNA fragment suitable for the assembly, plasmid pTB9l7 was digested with BspM\, treated with DNA polymerase-l large fragment, and finally digested with BamHI. The excised fragment, lacking the initiation methionine-coding region, was integrated into the vector fragment to generate expression plasmid pTB975.

struct plasmid pTB975 (Fig. 1). The host strain bearing a regulatable T7 RNA polymerase gene was established by transduction and transformation of E. coli MM294 with phage DE3 (14) and plasmid pLysS (14), followed by transformation with plasmid pTB975. Upon induction with isopropyl /3-D-thiogalactoside, the cells synthesized haFGF. Crude bacterial extract, prepared from a 1 -liter culture, was first applied to a heparin-Sepharose affinity column. Elution with 1.5 M NaCI-containing buffer showed two peaks on the chromatographic profile (Fig. 2A). The second peak, analyzed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, contained mainly 16-kDa protein immunologically reactive to anti-aFGF polyclonal antiserum (data not shown). Fractions in the second peak were then subjected to phenyl-Sepharose column chromatography to remove minor contaminants. In the presence of 1 M (NH4)2SO4 most of the loaded proteins were adsorbed to the resin. The proteins were eluted from the column with a concentration gradient of (NH4)2SO4, exhibiting a broad peak, with a minor one at its shoulder (Fig. 2B). The final material was obtained by collecting

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871

Characterization of human aFGF

1.5

~

1.0 •-0.75

E c o •-0.5

4

U re

00

•0.25

o V)

5 10 Fraction Number

6.5

15

50

60

Fraction Number

,,. ^^^

***

n \f

| 10

| \ 1 > 20 30 40 50 Retention Time (min)

60

Fig. 2. Purification of haFGF A, Crude extract prepared from the bacterial cells was loaded onto a heparin-Sepharose column. The column was washed with 0.5 M NaCI in 20 mM Tris-HCI (pH 7.4). Elution was accomplished with 1.5 M NaCI-containing Tris buffer and was monitored by the absorbance at 280 nm. Every 6 ml eluate were collected. B, Peak fractions (fractions 8-12) of the heparin-Sepharose column eluate were combined and applied to a phenyl-Sepharose column in the presence of 1 M (NH4)2SO4. The column was washed with 1 M (NH4)2SO4-20 mM Tris-HCI (pH 7.4). Elution was performed using a concentration gradient of (NH4)2SO4 ranging from 1 to 0 M in Tris buffer and was monitored by the absorbance at 280 nm. Every 2.5 ml effluent was collected. C, Purified haFGF was analyzed on SDS-PAGE, followed by Coomassie blue staining. D, Purified haFGF was applied to a reverse phase C4 HPLC column and eluted using a linear concentration gradient of acetonitrile ranging from 0-90% in 0.1% trifluoroacetic acid over 60 min at a flow rate of 1 ml/min.

the main peak while avoiding the shoulder peak portion. This material was electrophoretically pure (Fig. 2C) and judged to be 96% homogeneous by C4 reverse phase HPLC (Fig. 2D). Analyses of the amino- and carboxylterminal sequences and of the amino acid composition of the peak fraction eluate from the C4 reverse phase column were coincident with those predicted from the cDNA sequence. The purification of haFGF is summarized in Table 1. The biological activity was measured for each step by mitogenic assay using BALB/c 3T3 cells. Heparin-Sepharose column chromatography was responsible for most of the purification, resulting in 4.9fold purification. Purification was completed by phenylSepharose column chromatography. The final total ac-

tivity was 1.96 x 1010 U, which showed a recovery of 66%. Purified haFGF displays a specific activity of 0.59

u/pg. Mitogenic Activity for Fibroblasts and Endothelial Cells Mitogenic activity for BALB/c 3T3 cells was measured by a [3H]thymidine incorporation assay in both the absence and presence of heparin (Fig. 3). Heparin in various concentrations enhanced the cellular DNA synthesis induced, and the optimum concentration was estimated to be in the range of 10-50 Mg/ml- Heparin at this concentration favorably potentiated the action of

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MOL ENDO-1990 872

Table 1. Purification of haFGF Total Protein (mg)a

Crude Extract Heparin Column Phenyl-Sepharose Column

261.9 43.2 33.4

SA

0.11 0.54 0.59

Total Activity (x1010U)

Recovery

2.97 2.35 1.96

100 79 66

8

The total amount of protein was determined by the dyebinding method. " The biological activity of each fraction was evaluated by the thymidine incorporation assay on BALB/c 3T3 cells performed in the presence of 50 Mg/ml heparin. One unit of haFGF was defined as the activity necessary to half-maximally stimulate the cellular response.

haFGF at concentration of 6.4-32 pg/ml, and in the presence of heparin at this concentration, approximately 10 pg/ml mitogen were sufficient to stimulate the cellular DNA synthesis half-maximally. Purified hbFGF in the concentration range of 6.4-32 pg/ml also showed mitogenic activity, even in the absence of heparin, but this mitogenic activity was somewhat inhibited by heparin (50 Mg/ml; Fig. 3B). Also, the mitogenic activity of haFGF for human umbilical vein endothelial (HUVE) cells was assayed by counting the number of cells. In the absence of heparin, haFGF barely stimulated the proliferation of HUVE cells (Fig. 4). Only a

1.28

6.4

32

160

haFCF (pg/ml)

800

weak proliferative response of HUVE cells was obtained with haFGF in concentrations up to 20 ng/ml. On the other hand, in the presence of heparin (50 iig/m\), haFGF in a concentration of 32 pg/ml apparently promoted the proliferation of HUVE cells. The maximal response was observed with 4 ng/ml haFGF. In this range of haFGF concentrations, a dose-dependent proliferation was observed up to 4 ng/ml, but 20 ng/ml mitogen were less effective in this assay with heparin. Angiogenic Activity The angiogenic activity of haFGF was examined on chick embryo chorioallantoic membrane (CAM). Ten nanograms of haFGF induced angiogenesis in 67% of the CAMs, and with 100 and 200 ng haFGF, 9 1 % of the CAMs exibited an angiogenic response (Fig. 5). Thus, it became evident that haFGF without heparin can act angiogenic in a dose-dependent fashion. However, with the addition of 1 n,g heparin, each dose of haFGF induced angiogenesis in 100% of the CAMs (Fig. 5), while heparin alone did not induce a positive response. The angiogenesis induced by haFGF and haFGF with heparin is shown in Fig. 6. When 200 ng haFGF were given with 1 ^g heparin, many more blood capillaries ran to the disc than in the case of haFGF alone. Such a potentiating effect was also recognized

1.28

6.H

32

160

hbFCF (pg/ml)

Fig. 3. Mitogenic Activity of haFGF and hbFGF for BALB/c 3T3 Cells A, A31 cells were seeded in a microtiter plate (2 x 104/well), grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% calf serum for 1 day, and then starved in 0.2 ml DMEM supplemented with 0.5% calf serum for the next 3 days. Upon addition of sample, 10 ^l (1/20th volume) of culture medium was replaced with 0 (O), 8 (•), 40 (•), 200 (•), 1000 (A), or 5000 (•) Mg/ml heparin dissolved in DMEM. Purified haFGF was diluted with DMEM supplemented with 0.5% crystalline BSA and 0 (O), 0.4 (•), 2 (•), 10 (•), 50 (A), or 250 (•) ^g/ml heparin and then added to wells at the indicated final concentration. Cellular mitogenic response was determined by measuring [3H]thymidine uptake. B, The mitogenic activity of hbFGF was analyzed as described above in both the presence (•) and absence (O) of heparin. Results in A and B represent the average of two independent determinations performed in duplicate. The SD was less than 14% of the mean value.

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873

Characterization of human aFGF

100

200

10 32

160 haFCF

800

4000

20000

(pg/ml)

Fig. 4. Mitogenic Activity of haFGF for HUVE Cells HUVE cells were seeded at a density of 5 x 103 cells/cm2 in 1 ml GIT medium containing 2.5% fetal bovine serum. The next day, 10 ^ DMEM (O) or 5 mg/ml heparin-containing DMEM (•) were mixed in, and 10 n\ haFGF sample were added to the culture medium. Four days later, viable cells were trypsinized and then counted with a Coulter counter. Experimental points represent the average of two duplicate determinations. The SD was within 10% of the mean value.

with heparin and hbFGF in a similar mode (data not shown). Circular Dichroism (CD) Spectra of haFGF Since heparin appeared to significantly potentiate the mitogenic activity of haFGF, we examined the possibility that heparin might affect the secondary and/or tertiary structure of the mitogen. CD spectra of haFGF were examined in both the presence and absence of heparin. Human aFGF displayed a far-UV CD spectral pattern typical for a polypeptide without a significant secondary structure (Fig. 7A). Interestingly, heparin did not affect the spectrum of haFGF. The near-UV CD spectra of haFGF was also not affected by heparin (Fig. 7B). The near- and far-UV CD spectra of hbFGF were also determined (data not shown) and found to be similar to those described by Fox et al. (15). No difference in CD spectra was observed whether heparin was present or absent (data not shown). Affinity for Heparin Acidic and basic FGF are characterized by their strong affinity for heparin. Purified haFGF and hbFGF were

haFCF

(ng)

Fig. 5. Angiogenic Activity of haFGF Polypropylene discs carrying 10,100, or 200 ng haFGF with (•) or without (•) 1 ^g heparin were grafted onto CAMs. Three days later, the number of CAMs showing angiogenesis were counted. The height of each bar represents the number of CAMs averaged from three independent experiments using 10-13 CAMs. The SD was within 10% of the mean value. The average of background (angiogenesis toward discs carrying no sample) did not exceed 10% of the total angiogenesis in all experiments.

analyzed for their heparin affinity on HPLC. Human aFGF was eluted from the column at the concentration of 1.1 M under the linear gradient of NaCI (0-2 M; Fig. 8A), while hbFGF was eluted at a concentration of 1.3 M under the same conditions (Fig. 8B). Affinity for Copper Human aFGF and hbFGF were analyzed for their precise copper affinity on HPLC. Human aFGF was eluted from the column at a concentration of 16 ITIM under the linear gradient of imidazole from 0-30 HIM (Fig. 9A), while hbFGF was eluted at a concentration of 12 ITIM under the same conditions (Fig. 9B). Stability of haFGF under Acidic Conditions The stability of the mitogenic activity of haFGF and hbFGF under acidic conditions (pH4) was studied. Human bFGF was virtually inactivated in 30 min, while haFGF retained 16% of its activity after 60 min (Fig. 10A). Thus, haFGF was proved to be more stable than hbFGF at pH 4. Heparin was shown to protect FGFs from inactivation by acid (8). We studied the capability

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MOL ENDO-1990 874

Fig. 6. Angiogenesis Induced by haFGF Polypropylene discs carrying salt in buffer (A), 200 ng haFGF (B), 1 M g heparin (C), or 200 ng haFGF and 1 M g heparin (D) were grafted onto the CAMs. These were then photographed 3 days later.

- -12 2

200 210 220 230

2U0

250

260 270 280

290 300 310

Wavelength (nm)

Fig. 7. CD Spectra of haFGF Far-UV (A) and near-UV (B) CD spectra of haFGF were measured in both the presence (0.5 mg/ml; heparin. Data are represented as the mean residue ellipticity.

•) and absence (—) of

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Characterization of human aFGF

875

A

0.1 -

2.0 1.5

_ 0.05 -

- 1.0 0.5 •

J

0

B

0)

u c n

JQ

o

2.0

0.1 -

U)

.0

1.5

0.05 -

1 1 10

20

30

40

- 0.5

50

60

Retention Time (min) Fig. 8. Heparin Affinity HPLC of haFGF and hbFGF Purified haFGF (A) or hbFGF (B) was applied to a heparin affinity HPLC column and eluted using a linear concentration gradient of NaCI ranging from 0-2 M in 10 mM Tris-HCI (pH 7.4) over 60 min at a flow rate of 1 ml/min.

of heparin to stabilize haFGF further. In the absence of heparin, the residual activity of haFGF decreased to less than 25% within 10 min, and after 2 h, most of the activity was lost (Fig. 10B). On the other hand, haFGF still had more than 45% of its activity after 60 min when heparin was present, and 30% of the activity still remained after 2 h (Fig. 10B). Thus, haFGF was stabilized in the presence of heparin against inactivation at pH 4.

DISCUSSION

We used the T7 phage RNA polymerase system (13, 14) to produce haFGF in E. coli, and supported by the selective high level expression of haFGF, we could purify the mitogen to near homogeneity in a large quantity. The final preparation was 33.4 mg sufficiently homogenous protein (Fig. 2 and Table 1). This appears to be the best efficacy of aFGF production in E. coli. The primary structure of our purified haFGF was identical to the sequence predicted by the cDNA, including the methionine residue derived from the initiation codon. This indicated that no proteolytic degradation occurred during the purification steps. When hbFGF was ex-

pressed in E. coli, removal of the initiation codondirected methionine was observed (12, 16, 17), but such a fine-tuned processing has not been observed in the case of recombinant aFGF (17,18). The mitogenic activity of haFGF was found to be fairly dependent on the presence of heparin. When 1050 Mg/ml heparin were present, haFGF stimulated DNA synthesis in serum-starved BALB/c 3T3 cells half-maximally at a concentration of about 10 pg/ml, while in the absence of heparin even 800 pg/ml haFGF could not achieve stimulation at the same level (Fig. 3). Thus, it was shown that heparin made haFGF approximately 100 times as potent for quiescent BALB/c 3T3 cells. Similarly, heparin capacitated haFGF, a very poor mitogen for HUVE cells by itself, to promote the proliferation of HUVE cells (Fig. 4). On the other hand, hbFGF did not require heparin for its biological activity (Fig. 3B) despite its higher affinity for heparin relative to haFGF (Fig. 8). Indeed, heparin was somewhat inhibitory (Fig. 3B). It is interesting to note that heparin affects these closely related molecules in an opposite way, and it seems paradoxical that haFGF, a heparin-dependent mitogen, showed a lower affinity for heparin than hbFGF, which has no heparin requirement for expressing biological activity. It has been shown that heparin

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MOL ENDO-1990 876

20

30

40

50

60

Incubation Time (min)

10

20

30

40

50

60

Retention Time (min) Fig. 9. Copper Affinity HPLC of haFGF and hbFGF Purified haFGF (A) or hbFGF (B) was applied to a copper affinity HPLC column and eluted using a liner concentration gradient of imidazole from 0-30 mM in 20 ITIM sodium phosphate (pH 7.2)-1.5 M NaCI at a flow rate of 1 ml/min. The fractions were subjected to two-site ELISA using either antiaFGF peptide antiserum (A) or anti-bFGF MAb78 (B) as the second ligand.

influences cell surface binding of FGFs (10, 19) mediated by receptors (high affinity sites) and heparin-like molecules (low affinity sites) (19, 20). In the case of bFGF, the addition of heparin has been reported to quench the low affinity binding to endothelial cells without significantly decreasing the high affinity binding and consequent stimulation of plasminogen activator synthesis (19). On the other hand, heparin has been reported to enhance the total cellular binding of aFGF (10). Since it seems reasonable that heparin can compete for low affinity sites also in the case of aFGF, heparin may enhance the high affinity binding of aFGF. Schreiber ef a/. (10) proposed that heparin induces a conformational alteration of aFGF which is recognized as change in immunological epitope exposure. Jaye ef a/. (21) argued, from their analysis using fluorescence spectroscopy, that heparin decreases the surface exposure of Trp107 of aFGF. However, in our analysis, no drastic conformational alteration of haFGF caused by heparin was observed by CD spectra determination. Recent studies implicated that the restricted region in the cationic carboxyl-terminal sequence of FGFs contributed to heparin binding (6, 22). These observations suggest that heparin is not likely to induce significant conformational change, such as cyclization of the FGF

10 20 30 Incubation Time (min) Fig. 10. Stability of haFGF under Acidic Conditions Human aFGF (O) or hbFGF (A) was incubated at 37 C in 50 mM sodium acetate (pH 4.0) at a concentration of 1 Mg/ml (A). Human aFGF was incubated at 37 C in 50 mM sodium acetate (pH 4.0) at a concentration of 1 M9/ml in either the presence (•) or absence (O) of heparin (B). At the indicated time, 50 n\ incubation mixture were removed and mixed with 10 M' 1 M Tris-HCI (pH 8.0) and 440 MI DMEM containing 0.5% crystalline BSA. These mixtures were further diluted 20-fold with DMEM supplemented with 0.5% crystalline BSA and 50 M9/ml heparin, then subjected to [3H]thymidine incorporation assay in the presence of 50 ng/m\ heparin. The residual activity of each sample was calculated and expressed as a ratio to the activity at time zero. Each point was averaged from three independent assays, performed in duplicate. The SD was lower than 10% of the mean value.

molecule, but is likely to induce some localized microstructural alteration in the aFGF molecule which seemed to be responsible for the heparin-induced increase in receptor affinity of aFGF, as proposed by Schreiber ef al. (10).

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Characterization of human aFGF

877

Heparin has been shown to protect both aFGF and bFGF from inactivation by heat and acid (5, 8) and from degradation by certain proteolytic enzymes (5-7). Although the affinity of haFGF for heparin was indeed lower than that of hbFGF (Fig. 8), such a protective manner of heparin could participate in specific potentiation of the biological activity of haFGF if haFGF was far more unstable than hbFGF. Mueller et al. (23) reported the half-life of aFGF's mitogenicity in culture conditions to be 2.5-3 h, much shorter than the incubation period (16 h) in our 3T3 mitogenic assay. However, repeated feeding of 3T3 cells with haFGF alone for several hours (up to 8 h) at 1 -h intervals could not produce the cellular response observed in the presence of heparin (data not shown). These observations could not be elucidated by only the protective effect of heparin against proteolytic degradation of aFGF. Another mechanism seems to underlie the heparin-induced potentiation of cellular growth triggered by haFGF. The concentrations of NaCI required to elute haFGF and hbFGF from a heparin-affinity HPLC column were 1.1 and 1.3 M, respectively (Fig. 8). Thus, aFGF possesses lower affinity for heparin than bFGF derived from bovine (24) or human sources. Seno et al. (22) implicated the positive charge of carboxyl-terminal sequence of bFGF in heparin-binding. The carboxyl-terminal of bFGF is more rich in basic residues than that of aFGF (22). This feature possibly contributes the higher affinity of bFGF for heparin relative to that of aFGF. In addition, we noticed that haFGF was eluted from the column as a single peak even if applied under nonreducing conditions (data not shown). In the case of hbFGF applied under such conditions, multiple elution peaks are observed, as previously described (12, 25). In the case of hbFGF, intermoleculer disulfide bridging has been suggested to be responsible for the heterologous chromatographic profile of heparin-affinity HPLC (25); however, such a cross-linking effect does not appear to exist in the case of haFGF. Moreover, neither dimeric nor trimeric haFGF was detected on SDS-PAGE under nonreducing conditions (data not shown), under which multimerization of bFGF has been reported to be apparent on the gel (15). A 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration study resulted in the detection of 2.94 mol free sulfhydryl groups/1 mol haFGF, suggesting that no disulfide bond was present in the haFGF molecule (data not shown).

with it (9). Heparin enhanced angiogenesis induced by haFGF (Figs. 5 and 6). It is probable that a heparinhaFGF complex facilitated the diffusion of this factor in a stable form. We studied the stability of FGFs at pH 4 and found haFGF to be more stable than hbFGF (Fig. 10A). This may imply that haFGF effectively functions in the wound healing process, which involves an acidic environment caused by inflammation. The protective effect of heparin for FGFs from inactivation by acid (8) was confirmed for haFGF (Fig. 10B). The affinity of FGFs for copper was shown by Shing (26) using a biaffinity column of heparin and copper; however, precise characterization of this affinity has been obscure. Using a copper affinity HPLC column and an enzyme-linked immunosorbent assay (ELISA) system, we could determine the concentration of imidazole that elutes FGFs from the column. Human aFGF was eluted at an imidazole concentration of 16 mM, while hbFGF was eluted at 12 mM. These results demonstrated that the affinity of aFGF for copper was relatively higher than that of bFGF. In this column chromatography it was not possible to monitor the elution profile of FGFs by the absorbance at 280 nm, although we could not determine the reason. The detection of FGFs by two-site ELISA was effectively carried out to make the elution profiles. Here we described the purification and characterization of haFGF produced in E. coli. Purified haFGF was active in vivo and very potent in vitro in the presence of heparin, as has been shown in the case of brainderived haFGF (27). Thus, the recombinant E. coli expression system proved to be useful in the production of functional haFGF as well as bovine aFGF (16, 17), bovine bFGF (17), hbFGF (12,15-17), and their analogs (15, 21, 25). Using recombinant DNA techniques, we can now obtain haFGF, hbFGF, and modified hFGFs in sufficient quantity. These materials will be useful in the further study of FGFs and will facilitate clinical trials in areas such as wound and ulcer healing.

Angiogenic activity of haFGF was observed in vivo without heparin, while haFGF could not promote the proliferation of HUVE cells in the absence of heparin. This angiogenic activity was equivalent to that of hbFGF, which promoted the proliferation of HUVE cells in the absence of heparin (data not shown). This is not extremely paradoxical, since angiogenin, lacking mitogenic activity for endothelial cells, is really an angiogenic factor (reviewed in Ref. 3). By way of analogy with angiogenin, haFGF may be an active chemotactic factor even in the absence of heparin for endothelial cells, or heparin-like molecules existing in vivo may potentiate the angiogenic function of haFGF by forming a complex

Construction of Expression System for haFGF

MATERIALS AND METHODS

The cDNA was synthesized chemically according to the sequence reported by Jaye et al. (28), but with convenient restriction sites. This cDNA was inserted into plasmid pUC18 (29) for amplification. The haFGF cDNA was subjected to SspMI digestion, followed by a fill-in reaction with DNA polymerase-l large fragment, and then digested with SamHI. Plasmid pET3-c (13) receiving Nco\ linker with the sequence of 5'CCATGG-3' at the Nde\ site was subjected to an Nco\ digestion/fill-in reaction, followed by BamHI digestion. These DNA fragments were ligated to generate the expression plasmid pTB 975 (Fig. 2). For this plasmid to function, E. coli MM294 lysogenized with phage DE3 (14) was used as the host after transformation with plasmid pLysS (14).

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MOL ENDO-1990 878

Cultivation of E. coli E. coli cells harboring plasmid pTB975 and pLysS were grown at 37 C in 1 liter Luria-Bertani medium ( 1 % Bacto-tryptone, 0.5% yeast extract, 1 % NaCI) supplemented with 35 ng/rx\\ ampicillin and 10 ng/m\ chloramphenicol. When the Klett value of the culture medium reached 170, isopropyl /3-D-thiogalactoside was added to a final concentration of 0.5 mM, and cultivation was continued for an additional 3 h. The cells were frozen in dry-ice-ethanol and stored at - 2 0 C. Purification of Recombinant haFGF Frozen cells were thawed, resuspended in 100 ml ice-cold lysis buffer (10 mM Tris-HCI, pH 7.4-10 mM EDTA-0.6 M NaCI10% sucrose-0.25 mM phenylmethylsulfonylfluoride) and then subjected to lysis with hen egg lysozyme added to a concentration of 0.5 mg/ml at 0 C for 1 h and then at 37 C for 5 min. This lysate was briefly sonicated and centrifuged to obtain the supernatant. This crude extract was diluted 3-fold with chilled buffer A (20 mM Tris-HCI, pH 7.4-1 mM EDTA) and then applied to a heparin-Sepharose (Pharmacia, Uppsala, Sweden) affinity column (2.5 x 4 cm) preequilibrated with buffer A containing 0.2 M NaCI. After washing the column with buffer A containing 0.5 M NaCI, bound materials were eluted with buffer A containing 1.5 M NaCI, and fractions of 6 ml were collected. Fractions 8-11 were combined, mixed with an equal volume of buffer A containing 2 M (NH4)2SO4, and then subjected to chromatography on a phenyl-Sepharose (Pharmacia) column (2.5 x 8 cm) at a flow rate of 0.5 ml/min. Proteins were adsorbed to the resin in the presence of 1 M (NH4)2SO4 and then eluted under a gradient of (NH4)2SO4 from 1-0 M for 200 min. All of these procedures were performed at 4 C. Reverse Phase HPLC Purified haFGF was applied to a reverse phase HPLC C4 column (0.5 x 20 cm; VYDAC, Hesperia, CA). Elution was accomplished under a linear gradient of acetonitrile ranging from 0-90% in 0.1% trifluoroacetic acid for 60 min at a flow rate of 1 ml/min. Analysis of Amino Acid Composition and Terminal Sequence

with phosphate buffer (20 mM, pH 7.2). Eluates were concentrated and then dialyzed against buffer B (20 mM phosphate buffer, pH 7.2-0.2 M NaCI) at 4 C. Protein concentration was determined spectrophotometrically using the extinction coefficient of 1.0 for 1 mg/ml haFGF at 280 nm and a 1-cm path length. For analysis in the presence of heparin, one ninth of a volume of 5 mg/ml heparin in buffer B was added to the protein solution. CD spectra were determined at room temperature on a Jasco model J-20A spectropolarimeter (Jasco, Easton, MD) equipped with a Jasco DP-50IN data processor. Measurements were carried out using cuvettes of 1 and 0.1 cm for near- and far-UV ranges, respectively. The data are expressed as the mean residue ellipticity, calculated using a mol wt of 15,969 and a total residue number of 141 for recombinant haFGF. Heparin Affinity HPLC Purified protein solution was appropriately diluted with 20 mM Tris-HCI (pH 7.4) to reduce the NaCI concentration to 0.2 M; dithiothreitol was added to a final concentration of 1 mM, then incubation was performed at 37 C for 30 min just before application to the heparin affinity HPLC column (Shodex AF pak HR-894, 0.8 x 5 cm, Showa-denko, Japan). Elution was performed using a linear gradient of NaCI concentration from 0-2 M in 20 mM Tris-HCI (pH 7.4) for 60 min at a flow rate of 1 ml/min. Copper Affinity HPLC One hundred micrograms of haFGF or hbFCF were applied to a copper affinity HPLC column (Shodex AF pak IA-894, 0.8 x 5 cm). Elution was performed using a liner gradient of imidazole (0-30 mM) for 60 min at a flow rate of 1 ml/min. FGFs were detected by two-site ELISA, using heparin as the first ligand, as described by Sato et al. (33). Polyclonal anti-aFGF peptide (Leu131-Asp140) antiserum (kindly provided by Dr. K. Kondoh; Chemical Research Laboratories, Takeda Chemical Industries, Ltd.) and monoclonal antibody MAb78 against bFGF (34) were used as second ligands to haFGF and hbFGF, respectively. Horseradish peroxidase reaction with ABTS (2, 2'-azino-di[3ethyl-benzthiazoline sulfonate (6)]; Vector Laboratories, Inc., Burlingame, CA) was monitored at 415 nm.

Acknowledgments Human aFGF purified by reverse phase HPLC was hydrolyzed with 5.7 N HCI at 110 C for 24 h in the presence of 4% thioglycolytic acid. The hydrolysate was analyzed using a Hitachi 835 amino acid analyzer (Tokyo, Japan). The aminoterminal amino acid sequence was determined by a gas phase protein sequencer (model 470A, Applied Biosystems, Foster City, CA). The carboxyl-terminal amino acid was determined by hydrazinolysis.

We wish to thank Dr. A. Kakinuma for encouragement, Dr. F. Studier for providing plasmids pET3c and pLysS and phage DE3, Dr. K. Kondoh for providing anti-aFGF peptide antiserum, Dr. E. Matsutani for providing HUVE cells, Dr. K. Sudo for performing the CAM assay, Mr. A. Fujishima for determining CD spectra, Mr. K. Kawahara and Ms. S. Nakagawa for analyzing amino acid sequence and composition, and Dr. T. Kurokawa for helpful discussions.

Preparation of hbFGF Human bFGF was extracted from recombinant E. coli and purified as described previously (12). Bioassays Mitogenic activity was assayed on mouse BALB/c 3T3 cells or HUVE cells as previously described (30, 31), but, where indicated, heparin was added to the sample and culture medium. Each assay was performed in duplicate. Angiogenic activity was monitored by the chick embryo CAM method (32).

Received December 28, 1989. Revision received March 5, 1990. Accepted March 5,1990. Address requests for reprints to: Tatsuya Watanabe, Biotechnology Research Laboratories, Research and Development Division, Takeda Chemical Industries, Ltd., 17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan.

REFERENCES

CD Spectra Purified haFGF was rechromatographed on a heparin affinity HPLC column as described below, but Tris buffer was replaced

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Characterization of human aFGF

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Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produ...
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