Stabilizing basic fibroblast growth factor using protein engineering

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 701-708

Vol. 151, No. 2, 1988 March 15, 1988

STABILIZIHGBASIC FIBROBLAST GROgTlt FACTOR USIIIGPROTEIHEI~IHEERIHG Hasaharu

Seno, Reiko $asada, ~4akoto Iwane, Katsuichi $udo*, Tsutomu Kurokava, Kumiko Ito, and Koichl Igarashi

Central

Biotechnology Laboratories and ~Biological Research Laboratories, Research Division, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka 532, Japan

Received January 19, 1988

SU~Y: Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable

in maintaining their a c t i v i t y .

These f a c t s suggest that t h i s

been successfully modified by protein engineering.

protein

has

® 1988 A c a d . . . . Press, Inc.

Basic fibroblast growth factor (bFfiF) was first isolated from bovine pituitary and identified

by its ability to promote the proliferation

widespread distribution of bFGF, its multifunctional

character

of fibroblasts

(1,2).

The

the wide range of cell types on which it acts,

and

have been revealed (3,4,5).

Recently

human

bFGF

complementary DNAs (cDNAs) were cloned by Abraham et al. (6) and by us (7).

We also

expressed

heparin

affinity

the

cDNA

column.

in E.coli and purified recombinant human Through

these

purification steps,

multiple forms caused by sulfhydryl binding (8).

bFGF

by

we noticed that

a

bFGF

has

This heterogeneity can be reduced

by substituting serine for each cysteine through site directed mutagenesis to prevent conformational

change

and

the stability can be

increased

without

changing

the

biological activities of bFGF.

MATERIALSAHDIETIIODS Site directed muta~enesis oil_human bFGF cDNA: The EcoRI-BglII portion of plasmid pTB669 (8), which encodes haman bFGF, was inserted into the multicloning sites of

Abbreviations:

FGF.fibroblast

cDNh,complementary DNA;

growth factor;

bFGF,basic f i b r o b l a s t growth factor;

BSA,bovine serum albumin; SDS,sodium dodecyl sulfate.

0006-291X/88 $1.50 70l

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 151, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bacteriophage M13mp8 (9). The s i n g l e stranded recombinant phage was used as the template in s i t e d i r e c t e d mutagenesis. The procedure of mntagenesis is described elsewhere (10,11). Four o l l g o n u c l e o t i d e primers were designed to change the c y s t e i n e codons a t p o s i t i o n s 26, 70 ,88 and g3 to s e r i n e codons (Table 1). Each primer was used independently to induce a s i n g l e mutation, and each r e s u l t i n g M13 phage was used to s u b s t i t u t e more than two c y s t e i n e codons with s e r i n e codons. At each mutation s t e p , the e n t i r e sequence of the gene was v e r i f i e d to confirm the generated mutations by dideoxy sequencing. From each r e p l i c a t i v e form of H13 phage having the mutations, the EcoRI-PstI fragment encoding modified bFGF was cut out and i n t e g r a t e d downstream of the E . c o l i t r p promoter in ptrp781 (12). F i n a l l y plasmids having the genes encoding f i f t e e n types of modified bFGF were prepared (Table 2) and used to transform E . c o l i M~94 (13). Preparation o£_cell extracts: Cultures of E.coli transformed by each plasmid were grown as described (8). The cells were harvested ; suspended in one-twentieth volume of buffer (20mH Tris-NCl (pH7.6), 10% sucrose, 0.2H NaCl, 100~g/ml hen egg white lysozyme and ImH Phenyl-methyl-sulfonyl-fluoride) ; incubated for I hour at ~C ; and t r e a t e d a t 3~C f o r 3 min. After a b r i e f s o n i c a t i o n , a bacterial cell extract was prepared by centrifugation. Heparin_ affinity HPLC: The extract prepared from 500-ml culture of E.coli was applied to a DEAE-cellulose column (DE52,2.5X8cm,Whatman) equilibrated with 20mM Tris-HC1 (pH7.6), 0.2H NaC1, lmH EDTA. The flow through f r a c t i o n was c o l l e c t e d , a p p l i e d to a heparin a f f i n i t y HPLC column (Shodex AF pak HR-894,0.8Xhcm,Showa-denko) and the p r o t e i n was e l u t e d with a l i n e a r g r a d i e n t of NaC1 (0.6-2H) as described (8). The amount of bFGF was estimated by Protein Assay (Bio Rad Labs) using bovine serum albumin (BSA) as a standard. Effect oj~_oxldatlon reauent: The peak fraction eluted from the heparin HPLC column was dialyzed against distilled water for 3 hours at 4]]. The protein was freeze-dried and resolved in 5 0 ~ Tris-HCl (pIS.5) and 0.2H NaCl. After BSA to I mg/ml and HgO. to 20 mM as an oxidation reagent were added, 200~g of the protein was incubated =a~ 37~ for 30 min. After the incubation, the solution was applied to the heparin HPLC column and eluted as described above. Bioassavs: Growth stimulation of mouse BALB/c3T3 cells and human endothelial cells isolated from umbilical vein were assayed as described previously (14,15). Angiogenesis activity was monitored on the chick embryo chorioallantoic membrane (16) grafting polypropylene disc (diameter 6.0mm) carrying 6.5 to 50.Ong of modified bFGF purified by heparin affinity HPLC. Effect_of_acidic solvents: bFGF or modified bFGF purified through the heparin HPLC column was diluted to lpg/ml with 50 mM Na-acetate (pH4.0) and incubated at 4~, 25~ or 37C. At the end of the incubation, the pH was adjusted to 7.0 and the solution was diluted to 100ng/ml of protein with phosphate buffered saline containing 0.5% BSA for the bioassay on mouse BALB/c3T3 cells described above.

RESULTS k]~) DI~CI]~ION four synthetic oligonucleotides as the mutation

Utilizing each

cysteine

mutagenesis structure

codon

primers independently,

in bFGF cDNA was changed to a serine codon

(10,11).

This

type

by

site

of mutation causes a minimal alteration

designed

the primers

to introduce or to eliminate

recognition sites which facilitated I).

to

of the protein because the substitution of an oxygen atom (serine)

sulfur atom (cysteine) prevents disulfied bond formation at the mutation also

directed

some

for

site.

the a We

restriction

enzyme

the detection of the mutant phage clones

(Table

The mutated DNA coding each type of modified bFGF was inserted into the plasmid

ptrp781

and

fifteen types of modified bFGF lacking sulfhydryl

radicals

originally

procedure

described

present in bFGF molecule were produced in E.coli (Table2). These previously

modified

bFGFs

(8), and

were processed by

the

purification

the specific elution pattern from the heparin HPLC column 702

was

Vol. 151, No. 2, 1988

B IO C H EMI C AL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1.

p o s i t i o n of Cys s u b s t i t u t e d

Synthetic oligonucleotide primers

sequence of oligonucleotide primer *

$

modified r e s t r i c t i o n enzyme s i t e

$

26

5' CGTrCTTGCTGTAGAGCCGCT3'

Rsa I

70

5' AACGATTAGCGCTCACTCC3'

Hae I I

88

5' GTAACAGACTTAGAAGCTAGT3'

93

5' TCGAAGAAAGACTCATCC3'

**

$

g

*

AI u I

*

H infI

Four oligonucleotide primers were designed to create serine codons for the codons of Cys26, Cys70, Cys88, and Cys93. Each mutation is also designed to create a new restriction enzyme site or to eliminate the site for ease of detecting the mutant phage clone. Each mutated nucleotide is indicated by an asterisk (*).

observed for each bFGF (Fig. l).

In most of the modified bFGFs,

the main peak

(PI

fraction) eluted from the heparin HPLC column was observed at a retention time around 20

minutes.

electrophoresis.

This

peak

fraction showed a single band on SDS

polyacrylamide

The mutant Cys->Ser26 showed the retention t i m e of P I ,

faster than 20 minutes (Fig.l).

Table 2.

Modified bFGF CS1 CS2 CS3 CS4 CS12 CS13 CS14 CS23 CS24 CS34 CS123 CS124 CS134 CS234 CS1234 bFGF

In addition,

a little

the mutant either Cys->Ser26 or Cys-

Modified bFGFs and their specific activities

Plasmid Substituted position pTB739 pTB742 pTB743 pTB744 pTB776 pTB779 pTB763 pTB762 pTB778 pTB777 pTB764 pTB780 pTB781 pTB782 p~765 pTB669

26 70 88 93 26,70 26,88 26,93 70,88 70,93 88,93 26,70,88 26,70,93 26,88,93 70,88,93 26,70,88,93

Specific activity 0.4 0.9 1.0 1.0 0.5 0.5 0.3 I.I 0.8 0.5 0.4 0.1 0.5 0.9 0.1 1.0

The plasmids t h a t produce f i f t e e n types of modified bFGFs were prepared and the modified bFGFs were p u r i f i e d by heparin a f f i n i t y HPLC. The s p e c i f i c a c t i v i t y of P1 fraction was determined by stimulating DNA synthesis in quiescent mouse BALB/c3T3 cells (14). The values are depicted as bFGF for 1.0 in each case. 703

gel

Vol. 151, No. 2, 1988

0.02L

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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40

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time

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F i ~ . l . Elution p r o f i l e s through heparin affinity HPLCof mxtifiedbFGFs. The e x t r a c t prepared from 50011 culture of each transformant was applied to heparin a f f i n i t y HPLC and eluted as described in the t e x t . a; bFGF (control), b; C$1, c; C$2, d; CS3, e; CS4, f;CSl4, g; CS24, h; CS12, i;CSl3, j;CS23, k; CS34, I; CS123, m; CS124, n; CS134, o; CS234, p; C$1234. The four peaks (PI, P2, P3, P4) recognized in the case of original bFGF (8) are indicated respectively.

704

Vol. 151, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

>Ser93 showed a rather dull and low pattern of elution (Fig. l ) . indicate

the

However,

loose

the

Cys->Ser26

or

weak binding of these types of modified bFGF to

heparin.

decreased s p e c i f i c a c t i v i t y of P1 was only recognized in the

(Table 2).

quadruple

These observations

mutants,

method using

the

Therefore

which

it

seems d i f f i c u l t to

purify

the

must have the mutation at position 26 or

heparin

affinity

column as

reported

triple

g3,

previously

mutant or

by the

(8).

The

differences among the amounts of modified bFGFs eluted from the heparin column seems to

be

caused

by

the

different affinities

of

these

bFGFs for

heparin;

p o s s i b i l i t y that each modified bFGFs is d i f f e r e n t l y s o l u b i l i z e d from E . c o l i .

the can not

be eliminated. Only

three

purified

types

through

(8)(Fig.I).

the

of

modified bFGFs and the original

purification

the

bFGF

were

heparin

efficiently

column reported

These three modified bFGFs showed the same s p e c i f i c a c t i v i t y as that of

the o r i g i n a l bFGF (Table 2). time

procedure using

CS23 showed a high and sharp peak at a

retention

of around 20 minutes on the heparin HPLC column even a f t e r being incubated with

an oxidation reagent such as H202,

but in CS2 and C83,

at a retention time of around 40 minutes (Fig.2).

some protein was detected

These minor peaks are P2 in

the

case of CS3 and P3 in the case of CS2, both of which are observed in the case of the original bFGF. conformation

This f a c t indicates that the contribution of Cys70 and Cys88 to the of

respectively. treatment

the

original

bFGF is

When a reducing agent,

with _~202'

reflected

in

the

peaks,

such as d i t h i o t h r e i t o l ,

These

results

most of the CS2 or CS3 modified bFGF was eluted as a indicate

that

P3,

was added a f t e r the

peak at 20 minutes (data not shown) as had been shown in the case of (8).

P2 and

single

original

the cysteine residues at 70 and

bFGF

88 are

not

responsible for the a f f i n i t y to heparin and that they are exposed to the surface the

bFGF molecule to form the i n t r a - or intermolecular sulfhydryl

bridging,

of

which

r e s u l t s in several heterogeneous peaks on heparin a f f i n i t y chromatography (8).

The

absence

the

of cysteine residues at both 70 and 88 protects the bFGF molecule

from

oxidization of the sulfhydryl residues. Westall et a l . in

reported that bFGF from bovine brain was inactivated by incubation

an a c i d i c solvent (17).

acidic conditions.

We have t e s t e d the s t a b i l i t y of

The modified bFGFs (C$2,

bFGF in 50mH sodium acetate (pH4.0)at 3 7 ° C .

modified bFGFs under

C$3 and CS23) were more s t a b l e

than

Especially C$23 maintained 50 percent

of i t s a c t i v i t y for at l e a s t 20 minutes in t h i s a c i d i c environment while the o r i g i n a l bFGF was modified

inactivated

in 10 minutes (Fig.

We then

examined

whether

bFGFs carry the same biological a c t i v i t i e s as those of the original

the e f f i c i e n t l y purified bFGFs (CS2, their

3).

on the chick embryo c h o r i o a l l a n t o i c membrane (Table

3).

angiogenic

The modified

bFGFs showed somewhat higher growth stimulating a c t i v i t y on endothelial c e l l s that of original type (data not shown).

bFGF;

C S 3 , CS23 and o r i g i n a l bFGF) were assayed for

a b i l i t y to stimulate the growth of endothelial c e l l s and for t h e i r

activity

these

than

The modified bFGFs at amounts of 50ng and 705

Vol. 151,No. 2, 1988

BIOCHEMICAL ANDBIOPHYSICALRESEARCHCOMMUNICATIONS Pl

P2 P3 P4

V

VV

V 2.0M

0.01

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0.01

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E O

o

I

0.03

,I

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I 0 20 Retention

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FIR.2. Effect of oxidation of modified bFfFs on the elutlon profiles of heparin affinity HPLC. After appropriate incubation with 20mM H209, 200pg of the P1 fraction

of each text. fraction vertical

12.5ng

showed a s l i g h t l y low angiogenic a c t i v i t y compared to t h a t of

bFGF, that

sample was applied to heparin affinity HPLC ancr'eluted as described in the a; bFGF, b; CS2, c;CS3, d; CS23. Each retention time of the peak that appeared in the purification of the original bFGF (8) is indicated by a arrow on the top.

but

at 25ng the a c t i v i t y was almost same (Table 3).

the

original

I t can be

concluded

the a c t i v i t i e s of the modified bFGFs on the growth of e n d o t h e l i a l c e l l s

and on

angiogenesis are conserved a t almost the same level as the a c t i v i t i e s of the o r i g i n a l bFGF. We

have

cysteine heparin

residues affinity

activities analyses

described

of

substitution

of

We have also described the elution profiles from stability

in

acidic

conditions

three highly purified modified bFGFs (CS2,

CS3

and

the

and

CS23).

a

biological These

biological

these residues mainly participate in sulfhydryl bridging which

heterogeneity the

by serine. column,

the

indicate that Cys70 and Cys88 of bFGF are not essential for its

activities;

@hen

here the modification of human bFGF by

induces

in the elutlon profiles through heparin affinity column chromatography.

amino acid sequence of the basic and acidic FGF (18,19,20) 706

are

compared,

Vol. 151, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,., I O O

,r

> ,r

4a u 50 >

e¢ 0 0

,

"r

T

i

i

6

i0

20

30

40

50

60

Incubation

time

(min.)

Fi~.3. Stability of modified bFGFs in a c i d i c condition. The samples were diluted with 50mM14a-acetate (pH4.0) and incubated at 37"C. After appropriate neutralization, they were assayed on mouse BALB/c3T3 cells. The relative activity is calculated as the specific a c t i v i t y at time 0 regarding 100%. CS23. See text in detail for other conditions.

only

Cys26 and Cys93 are conserved,

considered activity

to

be

0;

bFGF, 4 ; CS2, m; CS3, O;

t h e r e f o r e the h e t e r o g e n e i t y in conformation is

a s p e c i f i c c h a r a c t e r of bFGF and

the

between these two p r o t e i n s may l i e in t h i s point.

c y s t e i n e r e s i d u e s are well conserved even in oncogenes, amino

acid

angiogenic cysteine family:

difference

homologies to FGF have been

residues

biological

Furthermore these

int-2,

h s t , and LS3,

reported r e c e n t l y (21,22,23)

function of these oncogene products.

in

two whose

implying

the

These f a c t s imply t h a t these

two

are e s s e n t i a l to the basic f u n ct i o n s of the p r o t e i n s in

the

a f f i n i t y f o r heparin and growth s t i m u l a t i n g a c t i v i t y on f i b r o b l a s t s .

Table 3.

Modified bFGF

50.0

CS2 CS3 CS23 bFGF vehicle

78 72 72 94 11

hngiogenesis activltie~

hngiogenesis activity (%) 25.0 12.5 44 61 56 50 6

22 17 22 33 11

6.25 ng 0 0 0 0 0

Angiogenesis activity was monitored on the chick embryo chorioallantoic membrane (16) grafting polypropylene discs carrying 6.25 to 50.0 ng of P1 fraction of modified bFGF. In each case, 18 embryos were used and the number of embryos on which the angiogenesis toward the disc was observed are presented as a percentage. 707

FGF

Vol. 151, No. 2, 1988

As the effectively antecedent conditions.

result

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

the

engineering oxidation

modifications described

bFGF.

This

here,

success demonstrates

and the s t a b i l i t y of i t s biological

These modified bFGFs are

expected to

application as accelerated remedies for injuries,

we have succeeded

in

bFGF's resistance

to

activity

under

be developed for

acidic clinical

burns and thrombi.

ACKNOWLEDGEMENTS: We thank Dr. A.Kakinuma for continuous encouragement; Dr. R.~rumoto for synthesizing the oligonucleotides; and Drs. Y.Kozai and E . ~ t s u t a n i for the biological assay. REF~ENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Gospodarowicz,D. (1974) Nature 249. 123-127. Gospodarowicz,D., Rudland,P., Lindstrom,J., and Benirschke,K. (1975) Adv.Metab. Disorders 8. 302-335. Esch,F., Baird,A., Ling,N., Ueno,N, HIII,F., Deneroy,L., Klepper,R., Gospodarowicz, D., Bohlen,P., and Guillemin,R. (1985) Proc.Natl.Acad.Sci.USA B2~ 6507-6511. Gospodarowicz,D. (1984) in: Mediators in Cell Growth and Differentiation (R.J.Ford and A.L.Maizel,eds.) ppi09-134 Raven, New York. Gospodarowicz,D., Mescher,A.L., and Birdwell,C.R. (1978) Natl. Cancer Inst. Monogr. 48. 109-130. Abraham,J.A., Vhang,J,L., Tumolo,A., Mergia,A., Friedman,J., Gospodarowicz,D., and Fiddes,J.C. (1986) EMBOJ. ~ 2523-2528. Kurokawa,T., Sasada,R., Iwane,M., and Igarashi,K. (1987) FEBS lett. 213. 189194. Iwane,M., Kurokawa,T., Sasada,R., Seno,M., Nakagawa,S., and Igarashi,K. (1987) Biochem.Biophys.Res.Commun. 146. 470-477. Messing,J., and Vieira,J. (1982) Gene, 19~ 269-276. Zoller,M.J., and Smith,M. (1983) Meth.Enzym. 100. 468-500. Winter,G., Fersht, A.R., Wilkinson, A.J., Zoller,M., and Smith,M. (1982) Nature 299. 756-758. M.Seno, Hinuma,S., Onda, H., and Igarashi,K. (1986) FEBS l e t t 199. 187-192. Backman,K., Ptashne,M., and Gilbert, W. (1976) Proc.Natl.Acad.Sci.USA ~ 41744178. Maciag,T., Cerundolo,J., I l s l e y , S . , Kelley,P.R., and Forand,R. (1979) Proc.Natl. Acad.ScI.USA ~ 5674-5678. Satoh,T., Kan,M., Kato,M., and Yamane, I. (1986) Biochem.Biophys.Acta. 887. 8693. Vu,M.T., Smith,C.F., Burger,P.C., and Klintworth,G.K. (1985) Lab. Inv. 52~ 499508. Westall,F.C., Rubin,R., and Gospodarowicz,D. (1983) Life Sci. ~ 2425-2429. Gimenez-Gallego,G., Rodkey,J., Bennet,C., Rios-Candelore,M., DiSalvo,J., and Thomas,K. (1985) Science 230. 1385-1388. Esch,F., Ueno,N., Baird,A., Hill,F., Denoroy,L., Ling, N., Gospodarowicz,D., and Guillemin,R. (1985) Biochem.Biophys.Res.Commun 133, 554-562. Jaye,M., Howk,R., Burgess,W., Ricca,G.A., Chiu,I., Ravera,M.W., O'Brien,S.J., Modi,W.S., Maclag, T., and Drohan,W.N. (1986) Science 233, 541-545. Dickson,C., and Peters,G. (1987) Nature 326. 833. Taira.M., Yoshida,T., Miyagawa,K., Sakamoto,H., Terada,M., and Sugimura,T. (1987) Proc.Natl.Acad.Sci.USA84. 2980-2984. BovI,P.D., Curatola,A.M., Kern,F.G., Greco,A., Ittmann,M., and BasiIico,C. (1987) Cell ~ 729-737.

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