WO1990011356A1

WO1990011356A1 - Expression vector, eukaryotic cells and method for the recovery of pdgf-aa

Info

Publication number: WO1990011356A1
Application number: PCT/EP1990/000440
Authority: WO
Inventors: Jürgen Hoppe; Wolfram Eichner
Original assignee: GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Priority date: 1989-03-17
Filing date: 1990-03-16
Publication date: 1990-10-04
Also published as: DE3908802A1; DE3908802C2

Abstract

The invention relates to expression vectors and a method for production of PDGF-AA in eukaryotic cells. The expression vector comprises a DNA sequence encoding PDGF-A, under the control of an SV40 promoter. The expression vectors are used in eukaryotic, particularly mammalian cells to produce PDGF in large quantities.

Description

Expression vector, eukaryotic cells and method for the recovery of PDGF-AA.

Platelet Derived Growth Factor 9PDGF0 is a principal mitogen in the serum which promotes the growth of fibroblasts and smooth muscle cells in vitro. In vivo, PDGF is stored in the alpha granules of the thrombocytes and released by stimulation of the thrombocytes. Highly purified PDGF is a basic protein which has considerable heterogeneity in its molecular weigth (Kb 27,000 to 31,000 d). The reasons for this heterogeneity are age, processing, and the existence of various isoforms of type AA, AB or BB. The biological activity of all these forms is destroyed by reduction of the disulfide bridges. Human PDGF from thrombocytes consists mainly of AB heterodimers; cf. (Heldin & Westermark 198 , Deuel et al. 1985, and Ross et al. 1986).

Amino acid sequencing in conjunction with DNA sequencing of the genes has shown that A and B are homologous. PDGF-B is virtually identical with the transforming gene product p28 of Simian Sarcoma Virus (SSV). In SSV-transformed cells corresponding homodimers of type BB have been detected, which displayed propert i es s i mi l ar to tho se of th rombocyti c PDGF . These BB d i mer s were secreted to only a small extent from the infected cells. PDGF-AA forms, however, were efficiently secreted by producing cells. There are an increasing number of indications that the three isoforms, AA, AB and BB, take care of different functions. For detailed research, therefore it was necessary to develop a new teaching whereby larger amounts of PDGF-AA can be obtained.

According to one embodiment of the invention, an expression vector is provided for the recovery of PDGF-AA with the following features:

- SV40 promoter and

- DNA range which is controlled by the said promoter and codes PDGF-AA of the following amino acid sequence:

SIEEAVPAVCKTRTVIYEIPRSQVDPTSAN FLIWPPCVEVKRCTGCCNTSSVKCOPSRVH HRSVKVAKVEYVRKKPKLKEVQVRLEEHLE

CACATISLNPDYREEDTDVR

or a DNA range of about the same number of base triplets which can be hybridized with the above DNA range.

The hybridizable DNA range can be hybridized at at least 20°C and especially at least 25°C and /or a sodium chloride concentration of 1M. The above-mentioned DNA Range can be synthesized by means of a DNA synthesizer or on a bioengi neering route as follows. According to a further embodiment of the invention, eukaryotic cells are provided for the recovery of PDGF-AA which are obtainable by chromosomal integration of the vector of the invention by the method of Wigler et al.; cf Cells, 16 (1979) 777-785. An example of a cell suitable for the integration is BHK-21

(ATCC-CCL 10).

According to an additional embodiment of the invention a method for the expression of PDGF-AA is provided, which is characterized in that

(al) a vector in accord with the invention is chromosomany integrated into a eukaryotic cell by the method of Wigler, Sweet, Sim, Wold, Pellicer, Lacy, Maniatis, Silverstein & Axel and

(a2) the cell obtained according to (al) is cultivated and PDGF- AA is expressed, or

(b) a eukariotic cell containing a vector according to the invention is cultivated and PDGF-AA is expressed.

BHK-21 (ATCC-CCL 10; U.S. Patent 4,049,494); Organon, OSS,

Netherlands; ECACC, Portandown Great Britain), for example, is suitable for the practice of the method of the invention.

PDGF-AA can, according to an additional embodiment of the invention, especially when it has been expressed by the method of the invention, can be processed from aqueous media, especially expression cultures, by adsorbing the PDGF-AA onto a ceramic material, such as glass, for example, especially porous glass.

In addition to the above-described processing, the adsorbed

PDGF-AA can be eluted and then subjected to a gel permeation chromatography and/or a reverse-phase HPLC chromatography. The i nventi on wi l l be expl ai ned be l ow by experi ment al d at a and 3 f i gures

F i g . 1

Construction of the pODa Expression Vector

An Rsal cleavage product of the c-DNS clone pPFa-1 which includes the entire precursor molecule of the PDGF-A chain was cloned "blund ends" into the Smal cleavage point of the SV40 expression vector pBEH where it is under the control of the SV40 "early" promoter and of the SV 40 "late" polyadenyl ati on sequence. The vectors are not represented in correct scale to one another.

Insert: Mature sequence of the rPDGF-AA expressed from BHK cells. A = alanine, D = aspartic acid, E = glutamic acid, F = phenyl- alanine, G = glycine, H = histidine, I = isoleucine, K = lysine, L = leucine, M = methionine, N = asparagine, P = proline, Q = glutamine, R = arginine, S = serine, T = threonine, V = valine, W = trypthophan, Y = tyrosine.

Fig. 2

Purification of rPDGF-A from supernatant liquors on cultures of transformed BHK CELLS (Subclone 39)

The samples were separated on a 15 % polyacryl amide gel in the presence of SDS and analyzed after Coomassie blue dyeing.

A = Conditioned culture supernatant liquor (fermentation)

B = Eluate CPG chromatography

C = Eluate P 100 chromatography

D = Purified PDGF-A after HPLC

E-H = Same application series as A-D, with the addition of 10 % mercaptcethanol (10', 100 °C) in each case.

Fig. 3

Biological activity of rPDGF-AA from BHK cells.

The detection of biological activity was performed by stimulating densely arrested mouse embryo cells (AKR-2B) for synthesis of DNA (Shipley et. al. 1984)

Live Material used Reference

SV40 Promoter and Vector

with SV40 Promoter Pharmacia pPFa-1 = pPFG-1 FEBS Lett., 223 (1987)

243-246 pBEH Gene, 68 (1988) 213-219 pAG60 AKR-28 Example for mouse fibroblasts BHK 21 ATCC-CCL 10 LTK

Material and Methods

Cells cultures supplied by Gibco. Chromatography materials:

Porous glass PG 120-200, Sigma; Bio-Gel B100, 200-400 mesh, Bio- Rad; modified silica gel 214 Tp 54, 10 μm (=Vydac C4) by Vydac (Macherey & Nagel); urea by Sigma. Ethylene glycol and buffers were from Merck. Restriction endonucl eases and ligases were ordered from Boehringer and BRL, respectively.

Analytic Methods

Growth promoting activity was analyzed according to Shipley et al. (1984) by determining the (³H) thymidine rate in density- arrested mouse AKR-2B fibroblasts. Protein content was determined according to Bradford. Ami notermi nal sequence analysis was performed by means of a gas-liquid phase protein sequenator (Applied Biosystems). PTH amino acids were determined "on line" by HPLC chromotography. Polyacryl amide gel electrophoresis in the presence of sodium dodecyl sulfate was performed as described (Hoppe et al., 1986). Standard DNA techniques were performed according to Maniatis (1982).

SUMMARY

An Rsal fragment 712 base pairs long from the vector pPFa-1 (= pPGF-1) (Hoppe et al. 1987), containing the range coding PDGF-A, was integrated into the Smal intersection of the SV40 expression vector pBEH (Artel et al. 1988) and thus brought under the control of the SV40 promoter. This plasmid was integrated with chromosomes stable into BHK21 cells by cotransfer with the plasmid pAG60 (Colbere-Garapin et al., 1988). After subclonation a cell line was obtained which secreted up to 1 mg/1 of PDGF-AA/day into the nutrient medium. PDGF-AA was able to be purified to homogeneity from supernatant fluids of these cell cultures by a three-stage process. The first step was adsorption onto porous glass, the second step gel permeation chromatography on Bio-Gel P100. The final purification was achieved by reverse-phase high- pressure liquid chromatography. The yields were approximately 0.2 mg PDGF-AA/1 of supernatant liquid from the cell culture. The specific activity was 4 ng/ml for 50% activation of the (³H) thymidine installed in AKR-2B mouse fi brobl asts

Example

Construction of the Expression Vector pODa

From the 1.2 Kb long Bam HI fragment of the plasmid pPFa-1 (Hoppe et al., 1987), which contains the complete coding range for

PDGF-A, a fragment 712 base pairs long was obtained by Rsal digestion, which likewise still contains the entire coding range. This fragment was integrated into the Smal intersection in the polylinker of the plasmid pBEH. The orientation was determined by SalI cleavage. A plasmid was obtained which contained the PDGF-A gene in the correct orientation under the control of the SV 40 promoter in the plasmid pBEH. This plasmid was named pODa.

Transfection of BHK21 cells

The transfection of BHK21 cells was performed substantially by the method of Wigler et al., 1979. The cells to be transfected were abtrypsinated in the exponential growth phase before reaching confluence, counted, reseeded in a cell density of 3 x 10⁵ cells per culture bottle (25 cm² bottom area) in 5 ml of culture medium and incubated for 24 h at 37°C. On the following day, 4 hours before adding the precipitates, the medium was removed and replaced with fresh culture medium. The precipitates were prepared as follows: 5 μg of the DNS (pODa) to be transformed were dissolved under sterile conditions together with 5 μg carrier DNS from LTK cells with 5 μg of selection DNS (pAG 60, Bam HI cut) in 0.25 ml of 250 mM CaCl₂. This solution was added slowly, drop by drop, with constant agitation by air injected through a cottonstuffed Pasteur pipette, to 250 μl of 2 x HEBS buffer (280mM NaCl, 50 mM HEPES, 1.5 nM NaH₂PO₃, pH 7.1), resulting in the formation of a DNS/calcium phosphate coprecipitate. The solution thus prepared was incubated for an another hour at room temperature for complete precipitation, and then added to the cultures to be transfected. After 16 h the medium was replaced with fresh culture medium. On the second day after addition of the precipitates selection began by replacing the medium with fresh culture medium to which 500 μg/ml of G 418 were added. The selection medium was renewed eve ry 2-3 days. After 2 to 4 weeks, 2000 colonies were recognizable. To select for a stable transformation the cells were given tow passages in selection medium. These colonies were combined and recloned by limit dilution. Some clones were tested for their productivity on PDGF-AA. A high producer (clone 39) was obtained.

Cultivation of Cells and Isolation of PDGF-AA

To obtain larger amounts of supernatant liquors from cell cultures, cells of clone 39 were cultivated in 15 roller bottles

(600 cm² area, Falcon) in HG-DMEM/10% FCS, 25 mM HEPES, pH 7.4 (37ºC). Afterreaching confluence the cells were twice washed with HG-DMEM, 25 mM HEPES, and kept in this medium for 2 days. Then they were cultivated for 12 hours in HG-DMEM, 25 nM HEPES, 10 % FCS. This procedure was carried on for 4 weeks. A total of about 25 liters of supernatant liquors were obtained. Proteins from the combined supernatant liquors were all absorged onto CPG 120-200 prorous glass. For this purpose, 1 % w/v of glass was added to the liquor and incubated for 1/2 h. CPG glass was collected in a column (5cm0) and the liquors were again filtered through the CPG glass. Then a washing was performed with 10 mM of sodium phosphate, 0.5 M NaCl buffer, pH 7.2, until the OD₂₈₀ (1cm) had dropped below 0.005. The elution of PDGF-AA was achieved with 0.4 M glycine/HCL, pH 2.0, 6 % urea, 6 % ethylene glycol. After the dialysis of fractions containing PDGF-AA against 10mM of sodium phosphate, pH 7.5, precipitation was performed by adding 80 % ammonium sulfate (saturation). The precipitate was dissolved in the smallest possible volume of 10mM acetic acid. 10 ml portions were applied to a column 2cm∅ x 140 cm (Bio-Gel P 100 in 0.1 M acetic acid. The rate of flow was about 15 ml/h. Fractions of 4 ml were collected. Aliquots of the fractions were analyzed by SDS gel el ectrophoresis. Fractions containing PDGF-AA were combined and placed on a Vydac C4 column (0.8 cm ∅ x 25 cm). The flow rate was 1 ml/min. The column had been balanced beforehand in 0.1 % trifluoroacetic acid. The elution was performed through a linear gradient of 14 % acetonitrile in 0.1 % trifluoroacetic acid/H₂O for 70 minutes at a rate of flow of 0.7 ml per minute. The extinction at 220 nm was continously recorded and fractions of 0.7 ml were collected. PDGF-AA eluted at an acetronitrile content of 26-27 % . Aliquots of the fractions were tested by gel electrophoresis and those containing no foreign protein were combined.

RESULTS

The transfection of BHK21 cells with the vector pODa yielded 2000 independent clones. By recloning, strains were obtained which differed greatly in the production of PDGF-AA. Clones were ob tained which under optimum conditions had as much as 1 mg/l of PDGF-AA in the supernatant liquor of the culture. One of these clones (clone 39) was further characterized.

By a simple cultivation procedure in roller bottles, 25 liters of supernatant liquor were obtained. The adsorption of the proteins on the porous glass proved to be optimal, both as regards the efficient reduction of a large volume of 25 1 to 250 ml, and as regards the theoretical yield of activity. A 5-fold concentration was achieved. By two additional chromatographic steps (gel permeation chromatography followed by reverse-phase high-pressure liquid chromatography) PDGF-AA was obtained in an approximately 50 % yield. About 0.2 mg of PDGF-AA was isolated from one liter of supernatant liquor. The material thus obtained is uniform in the SDS gel eletrocphoresis (Fig. 2) and in the aminoterminal sequence analysis it showed only the expected sequence SIEE. It is accordingly identified unequivocally as PDGF-AA. This material stimulated the installation of (³H) thymidine in densely arrested mouse fibroblasts AKR-2B semimaximal at a concentration of 5 ng/ml.

LITERATURE

Artelt, P., Morelle, C, Ausmeier, M., Fritzek, M., and Ηauser, H. (1988) Gene 68 , 213-219. Bradford, M. J. (1976) Anal . Biochem. 72, 248-253.

Colbere-Garapin et al . (1981) J. Mol . Biol . 150, 1-14.

Hoppe, J., Gatti, D., Weber, H. and Sebald, W. (1986) Eur. J. Biochem. 155, 259-264.

Hoppe, J. Schumacher, L. , Eichner, W. and Weich, H. A. (1987) FEBS Lett. 223, 243-246.

Maniatis, T., Fritsch, E . F. and Sambrook, J. (1982) in:

Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York.

Shipley, G. C, Childs, C. B., Volkenant, M. E. and Moses, H. L, (1984) Cancer Res. 44, 710-716

Wigler, M., Sweet. R., Sim, G.K. , Wold, B., Pellicer, A. , Lacy, E., Maniatis, T., Silverstein, S. and Axel, R. (1979) Cell 16, 777-785

PURIFICATION OF rPDGF-A FROM SUPERNATANT LIQUORS

(PROCESSING SCHEDULE)

24,7 l Conditioned Medium

at 4 °C applied to

250 ml Controlled pore glass

Wash with 10 mM sodium phosphate buffer (pH 7.1), 0.5 M NaCl, elute with 0.4 M glycine/HCl (pH 2.0), 6% urea, 6% ethylene glycol, dialyze against 10 mM sodium phosphate buffer (pH 7.5)

9.2 mg Ammonium sulfate precipitation

Add ammonium sulfate (80% saturation), dissolve sediment in 10 mM acetic acid, centrifuge and discard the pellet, reduce volume in vacuum concentrator

6.4 mg Gel filtration

Chromatograph on P-100 column, combine fractions containing PDGF

5 mg Reversed-phase HPCL

Chromatograph on Vydac C 4 HPLC column

Claims

C l ai ms

1. Expression vector for obtaining PDGF-AA with the following features:

- SV40 Promoter and

- DNA range which is controlled by the said promoter and codes PDGF-A of the following amino acid sequence:

SIEEAVPAVCKTRTVIYEIPRSQVDPTSAN FLIWPPCVEVKRCTGCCNTSSVKCOPSRVH HRSVKVAKVEYVRKKPKLKEVQVRLEEHLE CACATISLNPDYREEDTDVR

or a DNA range of approximately the same number of base triplets which can be hybridized with the said DNA range.

2. Expression vector of claim 1, characterized in that the hybridizable DNA range is hybridizable at at least 20 and especially at least 25°C and/or a sodium chloride concentration of 1M.

Eukaryotic cells for the recovery of PDGF-AA, obtainable by chromosomal integration of the vector of claim 1 or 2 by the method of Wigler, Sweet, Sim, Wold, Pellicer, Lacy, Maniatis, Silverstein & Axel.

Cells of claim 3, characterized in that the vector is integrated in BHK-21 (ATCC-CCL 10).

Method for the expression of PDGF-AA, characterized in that (al) a vector of claim 1 or 2 is integrated chromosomally into a eurkarytic cell by the method of Wigler, Sweet, Sim, Wold, Pellicer, Lacy, Maniatis, Silverstein & Axel, and (a2) the cell obtained according to (al) is cultivated and PDGF-AA is expressed, or

(b) a eurkariotic cell of claim 3 or claim 4 is cultivated and PDGF-AA is expressed.

Method of claim 5, steps (al) to (a2), characterized in that BHK-21 (ATCC-CCL 10) is used.

Method for the processing of PDGF-AA, especially that obtained in accordance with claim 5 or 6 from aqueous media, especially expression cultures, characterized in that PDGF- AA is adsorbed onto a ceramic material.

Method of claim 7, characterized in that glass, especially porous glass, is used as ceramic material.

9. Method of claim 7 or 8, characterized in that additionally the adsorbed PDGF-AA is eluted and then subjected to a gel permeation chromotography and/or to reverse-phase HPLC chromotography.