Journal of Biotechnology, 22 (1992) 299-310

299

© 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

BIOTEC 00705

Cloning and high level expression of a synthetic gene for human basic fibroblast growth factor Peter V. Milev, Oleg I. Georgiev, Plamen O. Tzarnoretchki and Asen A. Hadjiolov Institute of Cell Biology and Morphology, Bulgarian Academy of Sciences, Sofia, Bulgaria (Received 13 May 1991; revision accepted 17 August 1991)

Summary A gene encoding human basic fibroblast growth factor has been chemically synthesized, cloned and expressed in Escherichia coli as a biologically active protein. The 465 bp gene was assembled by enzymatic ligation of 6 pairs of oligonucleotides and cloned in the expression vector pLCII downstream from the strong PL promoter. This promoter directed the synthesis of a fusion protein between a 31 aminoacids fragment of the lambda phage clI protein and bFGF. A four aminoacid recognition sequence for the site-specific protease fXa was introduced in the plasmid construct and this allowed cleavage of the fusion protein at the boundary between clI and bFGF. bFGF was purifed close to homogeneity using a Heparin-Sepharose column and Mono S cation exchange chromatography. The use of the pLCII expression system resulted in the accumulation of 20 to 25 mg of purified bFGF per 1 of bacterial culture. The recombinant bFGF was mitogenic for mouse 3T3 fibroblasts and the dose-response curve was similar to the one for native bFGF. Recombinant DNA; Fusion protein; Mitogen; 3T3 cell

Correspondence to: A. Hadjiolov, Centre de Biochimie et de G6n6tique Cellulaires du C.N.R.S., 118 Route de Narbonne, 31062 Toulouse Cedex, France.

Abbreviations: bFGF, basic fibroblast growth factor; PMSF, phenylmethylsulfonyl fluoride; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; fXa, activated blood coagulation factor X [EC 3.4.21.6]; FCS, fetal calf serum; aa, aminoacids; m, molecular mass; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylenediamine tetraacetic acid disodium salt.

300 Introduction

Basic F G F is a heparin-binding protein of 155 aa (m =-18 kDa) with pI 9.6 which has been purified from many tissues: brain, pituitary, retina, kidney, myocardium and tumors: breast and bladder carcinoma, hepatoma, and glioma (Gospodarowicz et al., 1987). It is a potent mitogen for a wide variety of cell types of mesodermal and neuroectodermal origin (Gospodarowicz et al., 1986). b F G F stimulates angiogenesis (Folkman and Klagsbrun, 1987), induces the accumulation of granulation tissue (Davidson et al., 1985), and stimulates wound healing (Sprugel et al., 1988). b F G F affects functionally all three fundamental cell types found in the nervous system, namely glial and endothelial cells, for which it is mitogenic and certain classes of neurons for which it is a neurotrophic agent (Walicke et al., 1986; Schubert et al., 1987). The mitogenic, chemotactic and differentiating effects of b F G F on the cells in the connective and bone tissues make it a potential therapeutic agent for treating poorly healing wounds and bone fractures (Lobb, 1988). As a neurotrophic agent it has clinical potential in the treatment of neurodegenerative disorders such as Alzheimer's and Huntington's diseases (Stopa et al., 1990; Faktorovich et al., 1990). Since little b F G F has up to now been available, it is desirable to produce sufficient quantities of recombinant protein to be able to study further its biological functions and for preclinical studies. Here we report the design and assembly of a synthetic gene for b F G F and its high level expression in E. coli.

Materials and Methods

DNA synthesis and gene assembly Oligonucleotides were synthesized by the phosphoroamidite method using an Applied Biosystems 380A synthesizer. Twelve oligonucleotides were synthesized ranging in size from 65 to 86 bases with an overlap between adjacent oligonucleotides of 8 to 10 bases (Fig. 1). The oligonucleotides were purified by electrophoresis on 15% polyacrylamide gels under denaturing conditions (Maniatis et al., 1982). The oligonucleotides for the upper strand were designated: A, B, C, D, E, F and those for the lower strand: G, H, I, K, L, M. The 5' and 3' ends of the gene were synthesized with added BamHI and SalI cohesive ends. All the oligonucleotides with the exception of A and M were kinased and the complementary oligonucleotides (30 pmoles each) were annealed to each other. A : G was ligated to B : H , C : I to D:K, E : L t o F : M for 1 h at 28 °C, 2 8 ° C and 37 °C, respectively. Then the pairs AB : G H and CD : IK were ligated (for 1 h at 33 ° C to produce ABCD : GHIK. ABCD : G H I K was joined to the EF : LM pair (for 1 h at 33 ° C) and thus the complete coding sequence for b F G F was assembled. 0.5 U of T4 D N A ligase was used in all the ligation steps. The final ligation mixture was incubated again with T4 D N A ligase at 15 ° C for 1 h and the 484 bp b F G F gene was purified by electrophoresis and elution from a 2% low melting temperature

301

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CAAG GAAATACACCAGTTGGTATGTGGCATTGAAACGAACTGGG C AG G T T C C T T T A T G T G G T C A A C C A T A C A C C G T A A C T T T GCT T GAC CC G T C 3' 5,t~ AT TATA 5' A A C T T G G C C C C A A A A C A G G A C C T G G G C AG AA AT A T T T G A A C C G N'~ 3' G G G T T T T GT C C T G G A C C C G TC T T T k~/ A C G ~/er 3' A G C T A T A C T T T T T C T T CC A A T G T C T G C T A A G A g 3 T T A A C [(3 5' T C G A T A T G A A A A A G A A G G T T A C A GA C G A T T C T ~CA A T T G ~CAGCT Hpol G C $oli Fig. 1. The oligonucleotides used for the assembly of the synthetic bFGF gene. Conservative substitutions were made in the nucleotide sequence to introduce new restriction sites (boxed); the corresponding nucleotides in the human gene are shown above and below the sequence.

a g a r o s e gel ( M a n i a t i s et al., 1982). T h e synthetic g e n e was c l o n e d as d e s c r i b e d u n d e r Results.

Bacterial strains and plasmids p U C 1 8 was u s e d for initial cloning into D H 5 a cells. T h e e x p r e s s i o n v e c t o r was p L C I I ( N a g a i a n d T h o r g e n s e n , 1984, 1987) w h i ch was p r o p a g a t e d in t h e host strain

302 QY13 (F-, laCam, trPam, BB'bio256N +, ci857, 6H, Sm r, recA). This vector and host strain were kindly donated by Dr. Peter K6nig, Inst. Cell Biol., Zurich. Standard procedures were used for plasmid purification, transformation, D N A extraction and sequencing (Maniatis et al., 1982; Sanger et al., 1977; Vieira and Messing, 1982).

Isolation of recombinant basic FGF Cell lysis Transformed QY13 cells were grown at 30 o C in 2YT medium with 5 0 / z g m l - 1 ampicillin until they reach an optical density of 1 at 600 nm. Then the temperature was quickly raised to 42 o C by swirling the flasks in a 70 o C water bath. The flasks were immersed in a 42 ° C water bath for 15 min and the cultivation was continued at 39 ° C for 2.5 h. The cells were centrifuged for 20 min at 9000 x g and the pellet (1 liter culture, 3.9-4 g wet weight) was resuspended in 12 ml lysis buffer (50 mM Tris p H 8.0, 1 m M E D T A , 25% w / v sucrose, 1 m M Dq-T, 1 mM PMSF). The ceils were lysed by the addition of 1 mg ml-1 lysozyme and kept on ice for 30 min. The viscous lysate was treated with 10/zg m l - 1 DNase I with the addition of MgC12 to 10 mM. After a 30 min incubation on ice, 50 ml detergent buffer was added (50 m M Tris p H 8.0, 1 m M E D T A , 100 m M NaC1, 1 m M D T r , 1 mM PMSF, 0.6% sodium deoxycholate, 1% Nonidet NP-40); the mixture was incubated for 30 min on ice and centrifuged at 9000 x g for 15 min. The pellet was washed 3 times with 1% Triton-X 100, 50 mM Tris p H 8.0, 1 mM E D T A , 100 m M NaC1, 1 mM DTT, 1 mM PMSF (Marston, 1987; Nagai and Thorgensen, 1987).

Preparation of bFGF from inclusion bodies The washed pellet was solubilized in 6 M guanidine HC1, 0.25 M DTT, 50 mM Tris p H 8.0, 10 mM E D T A at a protein concentration of 4 mg m l - 1 and incubated for 2 h at 50 ° C. The solution was diluted slowly with three volumes of 50 mM Tris p H 8.0, 10 mM E D T A with simultaneous slow cooling to 20 ° C and then dialysed against 0.4 M NaC1, 10 m M Tris p H 7.5, 1 mM DTT, 1 m M PMSF (Knoerzer et al., 1989).

Purification of the solubilized bFGF The dialysate was applied directly on a Heparin-Sepharose column (Pharmacia), equilibrated with 0.4 M NaCI, 10 mM Tris p H 7.5, 1 mM PMSF, 1 m M DTT. A linear gradient from 0.4 M to 2.5 M NaC1 was used. Fractions were analysed by SDS-PAGE. The recombinant fusion protein (m = 23 kDa) eluted as a single peak from 1.5 to 1.8 M NaC1. The peak fractions were desalted on a PD10 column (Pharmacia) and adjusted to 0.1 M NaC1, 10 m M Tris p H 7.5, 1 m M PMSF, 1 m M DTT. These fractions were applied on a Mono S cation exchange column (Pharmacia) and eluted with a 0.1 to 1.2 M NaC1 gradient. There was a sharp peak at 0.66 M NaC1 containing the highly purified 23 kDa fusion protein. Protein concentrations at various stages of purification were determined by the Bradford assay (Bradford, 1976).

303

Cleavage of the fusion protein The peak fractions from the Mono S chromatography were desalted on a PD10 column and adjusted to 100 mM NaC1, 50 mM Tris pH 8, 1 mM CaC1. The protein solution (1 mg ml 1)was incubated with the factor Xa protease (Boehringer) for 3 h at 25 ° C with a substrate to enzyme ratio 200/1 (w/w) (Nagai and Thorgensen, 1987). The cleaved fusion protein was subjected to Heparin-Sepharose chromatography to remove the cII fragment, the 1.5-1.8 M NaC1 peak fractions were dialysed against water and lyophilized on a Speedvac evaporator. Bioassays Purified recombinant b F G F and native bovine b F G F were assayed for mitogenic activity on 3T3 fibroblasts (Swiss 3T3 and 3T6). Bovine brain b F G F ( R & D Inc.) was a gift from Dr. Wilfrid Seifert, Max Planck Institute, G6ttingen. Cells were seeded into 96-well microtiter plates in D M E M with 10% fetal calf serum (3 × 103 cells in 200 /xl per well). The cells were incubated for 7 d at 37 ° C. The spent medium was removed and replaced with 200 /xl per well of fresh DMEM, containing 0.3% FCS and various dilutions of recombinant and native bFGF. After 12 h 1 /zCi [3H]thymidine per well was added, dissolved in 50 txl D M E M plus 0.3% FCS. The cells were incubated for 24 h, the medium was removed, the wells washed once with phosphate buffered saline, and incubated twice for 15 min with cold 5% trichloroacetic acid on ice. The wells were dried and the precipitate was solubilized in 200 Izl of 0.3 N N a O H and counted in a Beckman liquid scintillation counter. Each dilution of b F G F was assayed in triplicate.

Results

The synthetic gene encoding the 155 aa form of human b F G F was designed on the basis of the known nucleotide sequence (Abraham et al., 1986). The precise sequence of the gene was based on the statistically preferred codons in highly expressed E. coli genes (Grantham et al., 1980) and new unique restriction sites were introduced in the sequence (see Fig. 1). The assembly of the b F G F gene was described in Materials and Methods. The assembled synthetic gene was introduced into the BamHI-SalI sites of pUC18 vector and propagated into E. coli D H 5 a cells. This construct was designated pUC18bFGF. The correct size and nucleotide sequence of the insert were verified by BamHI + SalI digestion and dideoxy sequencing. To express the b F G F protein in E. coli we used the pLCII expression vector (Nagai and Thorgensen, 1984, 1987). This vector was chosen because it directs the expression of a fusion protein between the N-terminal 31 aa of lambda phage cII protein (Schwarz et al., 1978) and the heterologous protein. This ensures higher stability of the eukaryotic proteins (Marston, 1987) and also high translational efficiency, because the phage sequence determines an appropriate structure of the m R N A in the vicinity of the ribosome binding site (McCarthy et al., 1986). The

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Fig. 2. Manipulations of the pLCITbFGF construct to obtain The trans|ationally effective plasmid pLCIIfXbFOF that directs the synthesisof a cleavable fusion protein.

305

hybrid gene is transcribed from the lambda PL promoter (Fig. 2). The host strain QY13 provides a thermosensitive repressor (ci857) so that protein synthesis from PL is completely repressed at 30 °C but can be induced at 42 ° C. This stringent control system avoids constitutive expression of the foreign protein which is often lethal to the bacterial cell. There is also a transcriptional terminator tR~ between the PL promoter and the gene; the host strain provides the lambda N antiterminator protein. If the sequence coding for the tetrapeptide IleGluGlyArg is introduced between the cII fragment and the foreign protein, the fusion protein can be cleaved with the site-specific protease blood coagulation factor Xa to obtain the authentic eukaryotic protein (Nagai and Thorgensen, 1984). The pLCII vector was digested with the restriction endonucleases B a m H I and SalI and ligated to the bFGF fragment obtained from pUC18bFGF by B a m H I + SalI digestion. This construct (pLCIIbFGF) was not a functional expression plasmid since the initiating codon for bFGF was not in frame with the cII protein. To overcome this the pLCIIbFGF construct was cleaved with NcoI (see Fig. 2), then blunted with mungbean nuclease. After treatment with B a m H I the plasmid had a B a m H I protruding end and a blunt end without the initiating ATG codon. The oligonucleotide pair 5,GATCCATCGAGGGTAGG 3, 3,GTAGCTCCCATCC 5, which has a 5 ' B a m H I compatible and a 3' blunt end and codes for the tetrapeptide IleGluGlyArg was then ligated to the modified plasmid. Thus, a translationaUy effective plasmid was obtained designated pLCIIfXbFGF (Fig. 2). The nucleotide sequence of this construct was checked by dideoxy sequencing using bFGF oligonucleotides as primers. Competent QY13 ceils were transformed with pLCIIfXbFGF and several colonies were used for small scale protein synthesis. The clones that gave highest expression of the 23 kDa fusion protein were chosen for large-scale protein synthesis, extraction and purification. The pLCIIfXbFGF vector directed the synthesis of a fusion protein that was insoluble and remained in the pellet after cell lysis and detergent washes. The fusion protein was in the bacterial inclusion bodies as is often the case with proteins expressed at a high level in E. coli. Such proteins are soluble under denaturing conditions. The pellet was solubilized in 6 M guanidine HC1 with 0.25 M DTT to prevent the formation of intermolecular disulfide bonds. For renaturation the protein was carefully diluted and dialysed to prevent aggregation to bFGF (Marston, 1987). The renatured bFGF was purified using Heparin-Sepharose affinity chromatography and Mono S cation exchange chromatography which is a modification of the procedures established for the purification of bFGF from bovine brain (Gospodarowicz et al., 1984; Gospodarowicz, 1987; Klagsbrun et al., 1987). The 23 kDa function protein was cleaved completely with fXa and authentic recombinant bFGF with m = 18 kDa was obtained (Fig. 3A). The purity of the preparation was more than 95% (Fig. 3B). Under nonreducing conditions of SDS-PAGE an additional band was observed (Fig. 3B) which probably represents a dimer form of

306

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Fig. 3. Expression and purification of bFGF protein. Proteins were analyzed by SDS-PAGE on 17.5% gels (Laemmli, 1970) and stained with Coomassie brilliant blue. (A) Lane 1, QY13 cells with pLCIIfXbFGF at 30 ° C; lane 2, QY13 cells with pLCIIfXbFGF after temperature induction; lane 3, bacterial proteins after detergent washes and solubilization with guanidine HCI; lane 4, clIbFGF fusion protein purified by Heparin-Sepharose chromatography; lane 5, eluate from the Mono S column; lane 6, recombinant bFGF after fXa digestion. Numbers on the left indicate the positions of molecular mass standards in kDa (Pharmacia). (B) Lanes 1-5, SDS-PAGE under reducing conditions. Lane 1, molecular mass standards (Pharmacia); lane 2, clIbFGF fusion protein; lanes 3-5, 4/xg, 8/~g, 16/xg of recombinant bFGF; lanes 6-8, 4 ~g, 8 tzg, 16 ~g of recombinant bFGF under nonreducing conditions.

bFGF. The dimer form was less than 10% of the purified protein, b F G F has 4 cysteines of which two probably exist in a free sulfhydryl form, while cysteines 26 and 93 form a disulfide bond (Fox et al., 1988). The dimer was probably formed by intermolecular disulfide bonds in spite of including the reducing agent D T T in most purification steps with the exception of fXa cleavage and lyophilization of the protein. Fox et al. (1988) also observed even more extensive dimer and tetramer formation in recombinant bFGF, although D T T was present in their purification protocol. In a typical experiment one litre of induced bacterial culture produced 3.9 g of cells (wet weight) and the total E. coil protein was 785 mg. As estimated by densitometry scanning of protein gels the recombinant fusion protein was 8% of the total protein or 62.8 mg and increased to 23% after the detergent washes. The yields of b F G F in different purification steps are shown on Table 1. With the pLCII expression system we produced 20 to 25 mg of purified recombinant b F G F per 1 1 bacterial culture which exceeds the yield of recombinant b F G F reported in most previously published methods (Iwane et al., 1987; Squires et al., 1988; Barr et al., 1988; Fox et al., 1988; Knoerzer et al., 1989). The recombinant b F G F was tested for its mitogenic activity on Swiss 3T3 and 3T6 mouse cells which are susceptible to b F G F (Gospodarowicz, 1987). The

307 TABLE 1 Purification of recombinant bFGF Purification steps

Yield in the step (%)

bFGF (mg)

Bacterial lysis and detergent washes Solubilization in 6 M guanidine HC1 and renaturation Dialysis Heparin-Sepharose chromatography Desalting on PD10 Mono S chromatography Desalting on PD10 Cleavage with fXa Heparin-Sepharose chromatography Dialysis against water and lyophilization

100 70 85 92 95 93 95 100 92 85

62.8 44 37.4 34.5 32.8 30.5 29 29 26.7 22.7

dose-response curve for the human recombinant bFGF favourably compares with that for native bovine bFGF (Fig. 4). The recombinant bFGF produced half-maximum stimulation of [3H]thymidine uptake at a concentration of 0.2 ng m1-1 of growth medium, whereas native bFGF produced half-maximum stimulation at 0.15 ng ml-1. At concentrations higher than 50 ng ml-1 both proteins caused lower stimulation of DNA synthesis as was previously reported for native bFGF (Fernig et al., 1990).

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308

Discussion Chemical synthesis and enzymatic assembly is a straightforward approach to the cloning and expression of genes with known nucleotide sequences. There are several strategies for the design and joining of the oligonucleotides to form the desired synthetic gene (Brousseau et al., 1982; Jay et al., 1984; Frank et al., 1987; Fox et al., 1988; Theriault et al., 1988; Knoerzer et al., 1989). In most approaches small oligonucleotides (10-15 bases) are synthesized, then ligated into larger blocks. These larger fragments are either ligated to form the synthetic gene (Jay et al., 1984; Frank et al., 1987) or cloned separately, then excised from plasmids, ligated and cloned again (Brousseau et al., 1982; Knoerzer et al., 1989). We chose a more direct approach: to synthesize longer oligonucleotides (65-86 bases) and to assemble the whole gene without subcloning of fragments. The synthetic gene was cloned without mutations and rearrangements which is in agreement with the results of Theriault et al. (1988) who found that the use of longer oligonucleotides is clearly an advantage in terms of ligation, purification of ligated products and the frequency of mutations, observed after cloning. We preferred to ligate the oligonucleotides stepwise to avoid rearrangements since T4 DNA ligase is known to catalyze ligation at mismatch overlaps and small gaps (Frank et al., 1987). We used higher temperatures in the ligation reactions to avoid unwanted pairings. The bFGF gene was designed incorporating codons that are preferentially used in E. coli but the high level of expression of bFGF could hardly be attributed to the differences in codon usage since there is growing evidence that codon usage does not play an important role in determining the efficiency of protein synthesis (Balbas and Bolivar, 1990). The high yield of recombinant bFGF is most probably due to the expression of bFGF as a fusion protein with a fragment of lambda cII protein. The cII protein is produced in great quantity in the early stage of lambda phage infection and the presence of its sequence and regulatory elements in the pLCIIfXbFGF construct ensures efficient and controlled translation initiation. The accumulation of the fusion protein in the inclusion bodies provides the advantage that it is protected from proteolysis and the inclusion bodies can easily be recovered by differential centrifugation of the cell lysate and detergent washes (Uhlen and Moks, 1990). The synthetic bFGF gene was successfully expressed in E. coli and biologically active bFGF was isolated in high yield and high purity. The availability of large quantities of recombinant bFGF should greatly facilitate future research and testing of this protein as a potential pharmaceutical.

Acknowledgements We express our gratitude to Prof. Walter Schaffner, Institute of Molecular Biology II, Zurich, for the synthesis of oligonucleotides at his Institute, to Dr.

309

Peter K6nig, Institute of Cell Biology, Zurich, for kindly providing the pLCII vector and QY13 host strain, and to Prof. Wilfrid Seifert, Max Planck Institute fiir Biophys. Chemie, G6ttingen for the gift of native bFGF.

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