NO176481B - Process for Preparation of a Fusion Protein - Google Patents

Abstract

Fusion proteins in which the C- or N-terminal essentially corresponds to the first 100 units of interleukin 2 are new. Also new are gene structures coding for the above fusion proteins, vectors containing these gene structures, and hot cells containing these vectors. Hirudin derivs. with an amino acide sequence begining N-terminally with Pro are new and claimed. Human interleukin 2 derivs. contg. Asp. C-terminally are new and claimed.

Description

The present invention relates to a method for producing a fusion protein, with a C- or N-terminal portion that corresponds to the first 90-132 amino acids of interleukin-2 (IL-2), but which does not exhibit any interleukin-2 activity.

In the genetic engineering of eukaryotic proteins, only a low yield is often achieved in bacteria, especially for small proteins with a molecular weight of up to 15,000 daltons, whose structures contain disulfide bridges. It is assumed that the proteins formed are rapidly degraded by host proteases. Gene structures that code for fusion proteins are therefore suitably constructed, whereby the unwanted part of the fusion protein is the host protein, which after the isolation of the primary product can be cleaved by methods known per se.

It has now surprisingly been found that an N-terminal part of interleukin-2, which essentially corresponds to the first 100 amino acids, is particularly suitable for the production of fusion proteins. The primary product is thus a fusion protein which is completely, or predominantly, made up of eukaryotic protein sequences. Surprisingly, this protein is obviously not recognized as a foreign protein by the relevant host organism, and is therefore not immediately degraded. A further advantage is that the functional proteins produced according to the invention are poorly soluble to insoluble, and can therefore easily be removed from the soluble proteins, for example by centrifugation.

Since according to the invention regarding the function as "ballast part" for the fusion protein it is not important that the interleukin-2 part constitutes a biologically active molecule, nor does it depend on the exact structure of the interleukin-2 part. It is sufficient that essentially the first 100 N-terminal amino acids are present. It is therefore possible, for example, to make variations on the N-terminus, which allow cleavage of the fusion protein if the desired protein is arranged N-terminal to this. Conversely, C-terminal variations can be made to enable or facilitate the cleavage of the desired protein if this, as usual, is bound C-terminally in the fusion protein.

The natural DNA sequence encoding human interleukin-2, hereinafter "IL-2", is known from the European patent publication EP-A1-0091539. The literature indicated there also relates to mouse and rat IL-2. This mammalian DNA can be used for the synthesis of the proteins produced according to the invention. However, synthetic DNA, particularly advantageous DNA for human IL-2, is more appropriately used, as proposed in the (not previously published) German specification 3419995 (corresponding to the European patent publication 0163249). This synthetic DNA sequence is reproduced in the appendix (DNA sequence I). This synthetic DNA part not only has the advantage that, in terms of codon selection, it is matched to the conditions in the most frequently used host, E.coli, but it also contains a number of cut sites for restriction endonucleases, of which, according to the invention, use can be made. In the following table 1, a selection of the suitable incision sites at the beginning, respectively in the area of the 100th triplet, is reproduced. However, it is not excluded that variations in the DNA are made in the intermediate area, whereby use can be made of the additional cut sites indicated in the above-mentioned patent publication.

If the nucleases Ban II, Sac I or Sst I are used, an IL-2 partial sequence is obtained, which codes for approx. 95 amino acids. This length is generally sufficient to obtain an insoluble fusion protein. When the heavy solubility, for example in the case of a desired hydrophilic eukaryotic protein, is still not sufficient, but one - in order to produce as little "ballast" as possible - does not want to make use of the cut sites that are closer to the C-terminus, then one can by means of a corresponding adapter, respectively connecting link, extend the DNA sequence at the N- and/or C-terminal end, and thus "tailor" the "ballast" part. One can of course also use the DNA sequence, more or less, to the end, and in this way obtain possibly modified, biologically active IL-2 as a "by-product", respectively produce a bifunctional protein that shows IL-2 action in addition to the effect of the encoded protein.

Accordingly, the invention provides a method for producing a fusion protein, with a C- or N-terminal portion corresponding to the first 90-132 amino acids of interleukin-2 (IL-2), but not exhibiting any interleukin-2 activity, with the the general formula where X is the amino acid sequence of the first 90-132 amino acids of human interleukin-2, Y means a direct link or bridge of 2-14 genetically coded amino acids, which enables cleavage of the amino acid sequence Z, and Z is a sequence of genetically coded amino acids , where Y, neighbor to Z, contains Met, Cys, Trp, Arg, Lys, Asp or Pro or consists of these amino acids. The method is characterized by transforming a host cell with a gene structure encoding this fusion protein, culturing the host cell and separating the expressed fusion protein from the soluble proteins, preferably by centrifugation.

As can be seen from the formulas Ia and Ib, and as already mentioned above, it is possible to bring the desired protein to expression before or after the IL-2 part. In order to achieve a simplification, the following essentially describes the first-mentioned possibility, which corresponds to the original method for the production of fusion proteins. When this "classic" variant is described below, the second option is not excluded.

The cleavage of the fusion protein can take place in a known manner chemically or enzymatically. The choice of the appropriate method depends above all on the amino acid sequence of the desired protein. When this, for example, does not contain methionine, Y can stand for Met, after which a chemical cleavage takes place with chlorine or bromine. If in the link Y cysteine is at the carboxy terminal or if Y stands for Cys, then an enzymatic cysteine-specific cleavage or a chemical cleavage can take place, for example after specific S-cyanylation. If in the bridge link Y tryptophan is at the carboxy terminal or Y stands for Trp, then a chemical cleavage with N-bromosuccinimide can take place.

Proteins which in the amino acid sequence do not contain

and are sufficiently acid-stable, can be proteolytically cleaved in a manner known per se. This results in proteins whose N-terminus contains proline, respectively C-terminus aspartic acid. In this way, modified proteins can also be synthesized.

The Asp-Pro bond can also be made acid-labile when this bridge link is (Asp)n-Pro or Glu-(Asp)n-Pro, whereby n is 1 to 3.

Examples of enzymatic cleavages are also known whereby modified enzymes with improved specificity can be used (see C.S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, then a peptide sequence where a trypsin-cleavable amino acid (Arg, light) is bound to the N-terminal amino acid (Phe) of proinsulin, for example Ala-Ser-Met-Thr- Arg, since the arginine-specific cleavage can take place with the protease trypsin.

If the desired protein does not contain the amino acid sequence

then the fusion protein can be cleaved with factor Xa (European Patent Publications 0025190 and 0161973).

The fusion protein is obtained by expression in a suitable expression system in a manner known per se. For this purpose, all known host-vector systems are suitable, i.e. for example mammalian cells and microorganisms, for example yeast types and preferably bacteria, especially E.coli.

The DNA sequence, which codes for the desired protein, is incorporated in a known manner into a vector, which ensures good expression in the chosen expression system.

In bacterial hosts, the promoter and operator are suitably selected from the group lac, tac, trp, Pj^ or Pjj for the phages X, hsp, omp or a synthetic promoter, for example as proposed in the German specification no. 3430683 (European patent publication 0173149). Advantageously, the tac promoter-operator sequence is now commercially available (eg expression vector pKK223/3, Pharmacia, "Molecular Biology, Chemicals and Equipment for Molecular Biology", 1984, p. 63).

When expressing the fusion protein by the method according to the invention, it may prove appropriate to change triplets for the first amino acids after the ATG start codon in order to prevent any base pair formation at the level of mRNA. Such changes, as well as changes, omissions or additions of certain amino acids in the IL-2 protein portion, are known to the person skilled in the art.

The invention is described in more detail in the following examples and in the figures.

Fig. 1 and its continuation, fig. 1a, relates to the synthesis of the plasmid pk360, which codes for a fusion protein, which exhibits the hirudin sequence,

fig. 2 and its continuation, fig. 2a, relates to the synthesis of the plasmid pK410, which also codes for a fusion protein with the amino acid sequence of hirudin,

fig. 3 and its continuation, fig. 3a to 3c, relate to the construction of plasmids pPH15, 16, 20 and 30, which encode fusion proteins containing the amino acid sequence of monkey proinsulin. Fig. 4 relates to the synthesis of the plasmid pPH100, which codes for a fusion protein with the amino acid sequence of hirudin. Fig. 5 and its continuation, fig. 5a, shows the construction of the plasmid pK370, which codes for a fusion protein with the amino acid sequence of hirudin, as well as

fig. 6 and its continuation, fig. 6a, relates to the synthesis of the plasmid pKH101, which encodes a fusion protein with the amino acid sequence of monkey proinsulin.

The figures are not rendered to the correct scale, above all in the rendering of the poly connection point the scale is "outstretched".

EXAMPLE 1

Upon introduction of the lac repressor (P.J. Farabaugh, Nature 274

(1978) 765-769) in the plasmid pKK 177-3 (Amann et al., Gene 25

(1983) 167) the plasmid pJF118 (1) is obtained (fig. 1, see German patent application P3526995.2, example 6, fig. 6). This is opened at the singular restriction site for Ava I and reduced in a manner known per se by exo-nuclease treatment with approx. 1000 bp. After ligation, the plasmid pEW 1000 (2) is obtained (Fig. 1), in which the lac repressor gene is complete, but which, due to the reduction, is present in a significantly higher copy number than the starting plasmid.

Instead of the plasmid pKK177-3, one can also start from the above-mentioned commercial plasmid pKK223-3, build in the lac repressor and shorten the obtained product in an analogous way.

The plasmid pEW 1000 (2) is opened with the restriction enzymes EcoR I and Sal I (3).

The hirudin-encoding plasmid (4), prepared according to German specification 3429430 (European patent publication 0171024), example 4 (fig. 3), is opened with the restriction enzymes Acc I and Sal I and the small fragment (5), which for the most part contains the hirudin sequence , is isolated.

The plasmid p159/6 (6), prepared according to the German specification 3419995 (European patent publication 0163249), example 4 (Fig. 5), is opened with the restriction enzymes Eco RI and Pvu I and the small fragment (7) is isolated, which contains the largest part of the IL-2 sequence. This partial sequence, and in the following also other abbreviated IL-2 sequences, are designated in the figures as "AIL2".

The sequences (3), (5), (7) and the synthetic DNA sequence (8, fig. 1a) are then treated with T4 ligase. The plasmid pK360 (9) is obtained.

Competent E.coli cells are transformed with the ligation product and plated on NA plates containing 25 pg/ml ampicillin. The plasmid DNA preclones are characterized using restriction and sequence analysis.

An overnight culture of E.coli cells containing the plasmid (9) is diluted with LB medium (J.H. Miller, "Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 50 µg/ml ampicillin, in a ratio of approximately 1:100 and the growth is monitored using 0D measurement. At OD = 0.5, the shaking culture is set to 1 mM isopropyl-e-galactopyranoside (IPTG) and the bacteria are centrifuged after 150 to 180 minutes . The bacteria are boiled for 5 minutes in a buffer mixture (7M urea, 0.1£ SDS, 0.1 M sodium phosphate, pH 7.0) and samples are applied to an SDS/gel electrophoresis plate. After electrophoresis, it is obtained from the bacteria containing plasmid (9), a protein band corresponding to the size of the expected fusion protein. After digestion of the bacteria ("French Press"; "Dyno-Miihle") and centrifugation, the fusion protein is in the sediment so that significant amounts of the other proteins can be separated with After isolation of the fusion protein, d an expected hirudin peptide freed by cyanogen bromide cleavage. The peptide is characterized after isolation by protein sequence analysis.

The stated induction conditions apply to shaking cultures: for larger fermentations, corresponding OD values and possibly slightly varied IPTG concentrations are appropriate.

EXAMPLE 2

The plasmid (4) (Fig. 1) is opened with Acc I, and the upstream ends are filled in with Klenow polymerase. It is then cut with Sac I and the fragment (10) is isolated, this contains the largest part of the hirudin sequence.

The commercially available vector pUC 13 is opened with the restriction enzymes Sac I and Sma I and the large fragment (11) is isolated.

Using T4 ligase, the fragments (10) and (11) are now ligated to plasmid pK 400 (12) (Fig. 2). The plasmid (12) is reproduced twice in fig. 2, whereby the amino acid sequence of the thus obtained hirudin derivative is highlighted in the bottom representation.

The plasmid (4) (fig. 1) is opened with the restriction enzymes Kpn I and Sal I and the small fragment (13) containing the hirudin partial sequence is isolated.

The plasmid (12) is reacted with the restriction enzymes Hine II and Rpn I and the small fragment (14) containing the hirudindel sequence is isolated.

The plasmid (9) (fig. 1a) is partially cleaved with EcoR I, the free ends are filled in with Klenow polymerase in a fill-in reaction, and cut with Sal I. The derivative of the plasmid pK360 (15) is obtained.

By ligation of the fragments (3), (13), (14) and (15), the plasmid pK 410, (16) is obtained, which is reproduced twice in fig. 2a, whereby the lower rendering shows the amino acid sequence of the fusion protein and thus the hirudin derivative obtained after acid cleavage.

After expression and processing according to example 1, a new hirudin derivative is obtained, which in positions 1 and 2 exhibits the amino acids proline and histidine. This hirudin derivative shows the same activity as the natural product according to the German Explanatory Document No. 3429430, which in these positions exhibits the amino acids threonine and tyrosine, but is more stable against attack by aminopeptidases, whereby advantages can be obtained in in-vivo use.

EXAMPLE 3

The commercially available vector pBR 322 is opened with Barn H 1, whereby the linearized plasmid (17) is obtained. The free ends are partially fulfilled using dATP, dGTP and dTTP, and the overlying nucleotide G is degraded with S1 nuclease, whereby the pBR 322 derivative (18) is obtained.

The Hae III fragment (19) of monkey proinsulin (Wetekam et al., Gene 19 (1982) 181) is ligated with the modified plasmid (18), whereby the plasmid pPH 1 (20) is obtained. As the insulin sequence was used in the tetracycline gene, the clones containing this plasmid are not resistant to tetracycline and can be identified on this basis.

The plasmid (20) is opened with Barn Hl and Dde I and the small fragment (21) is isolated.

In addition, the Dde I-Pvu II subsequence (22) is isolated from the monkey proinsulin sequence.

The vector pBR 322 is opened with Bam HI and Pvu II and the linearized plasmid (22) is isolated.

By ligation of the insulin partial sequences (21) and (22) with the opened plasmid (23), the plasmid pPH5 (24) is obtained. This is opened with Barn Hl and Pvu II and the small fragment (25) is isolated.

To complete the insulin structure, the DNA sequence is synthesized (26).

The commercially available vector pUC 8 is opened with the enzymes Bam HI and Sal I and the residual plasmid (27) is isolated. This is ligated with the DNA sequences (25) and (26) to plasmid pPH 15 (28). This opens with Hall I and the above ends are fulfilled. From the resulting plasmid derivatives (29), the DNA sequence (30) is cleaved with Bam H1.

The commercially available vector pUC 9 is opened with the enzymes Bam HI and Sma I and the large fragment (31) is isolated. This is ligated with the DNA sequence (30) whereby the plasmid pPH16 (32) is obtained.

The plasmid (32) is opened with Sal I and the linearized plasmid (33) is partially filled with dCTP, dGTP and dTTP and the remaining nucleotide T is cleaved with Sl-nuclease. The resulting plasmid derivative (34) is treated with Bam HI and from the product (35) the overlying single strand is removed with Sl-nuclease, whereby the plasmid derivative (36) is obtained.

The blunt ends of the plasmid derivative (35) are ring-closed to plasmid pPH 20 (37).

Competent E.coli Hb 101 cells are transformed with the ligation mixture and plated on selective medium. Clones containing the desired plasmid express proinsulin, of 70 clones examined, 26 contained radioimmunologically detectable proinsulin. The plasmids are further characterized by means of DNA sequence analysis. They contain DNA, which in front of the codon for the first amino acids of the B chain (Phe) codes for arginine. The plasmid (37) is cleaved with Hind II, the ends above are filled in and subsequently cleaved with Dde I. The small fragment (38) is isolated.

The plasmid (28) (fig. 3a) is cleaved with Sal I and Dde I and the small fragment (39) is separated.

The plasmid (9) (fig. 1a) is first cleaved with Acc I, the free ends are filled in and then partially cleaved with Eco RI. The fragment (40) containing the shortened IL-2 sequence is isolated.

The linearized plasmid (3) (fig. 1) and the DNA segments (38), (39) and (40) are then ligated to the plasmid pPH 30 (41). This plasmid codes for a fusion protein which, in addition to amino acids 1 to 114 of IL-2, has the following amino acid sequence:

The arginine as the last amino acid of this bridge link Y enables cleavage of the insulin chain with trypsin.

Starting from plasmid (9) (fig. 1a), one can also arrive at plasmid (41) by the following procedure: One opens (9) with Acc I, completes the ends above, cuts with Sal I and ligates the resulting plasmid derivative ( 42) with segments (3), (38) and (39).

EXAMPLE 4:

The plasmid (6) (fig. 1) is opened with the restriction enzymes Taq I and Eco RI and the small fragment (43) is isolated. This fragment is ligated with the synthesized DNA sequence (44) and segments (3) and (5) of plasmid pPH 100 (45 ). This plasmid codes for a fusion protein, whereby the first 132 amino acids for IL-2 are followed by the bridge link Asp-Pro and then the amino acid sequence of hirudin. The proteolytic cleavage consequently yields a modified, biologically active IL-2', in which position 133 contains Asp instead of Thr, and a hirudin derivative which N-terminally contains Pro before the amino acid sequence of the natural product. This product is also biologically active, and compared to the natural product more stable against attack by proteases. The IL-2' hiru fusion protein also exhibits biological activity: In cell proliferation experiments with an IL-2-dependent cell line (CTLL2), biological activity was found.

After denaturation in 6 M guanidinium hydrochloride solution and subsequent renaturation in buffer solution (10 mM Tris-HCl, pH 8.5, 1 mM EDTA), high IL-2 activity was further found. Furthermore, the coagulation time of acid-treated blood mixed with thrombin was prolonged after addition of the fusion protein.

Consequently, a bifunctional fusion protein is obtained.

EXAMPLE 5:

The commercially available vector pUC 12 is opened with the restriction enzymes Eco RI and Sac I. In this linearized plasmid (46) an IL-2 partial sequence is inserted, which has been cleaved from the plasmid (6) (Fig. 1) with the restriction enzymes Eco RI and Sac I. This sequence (47) comprises the complete triplet for the first 94 amino acids of IL-2. By ligation of (46) and (47), the plasmid pK 300 (48) is obtained.

The plasmid (9) (fig. 1a) is opened with Eco RI, the upstream ends are filled in and post-cleaved with Hind III. The small fragment (49) is isolated, which in addition to the DNA sequence coding for hirudin contains part of the polyfor link from pUC 12.

The plasmid (48) is opened with the restriction enzymes Sma I and Hind III and the large fragment (50) is isolated. By ligation of (50) with (49), plasmid pK 301 (51) is obtained.

Competent E.coli 294 cells are transformed with the ligation mixture. Clones containing the plasmid (51) are characterized by restriction analysis. They contain DNA which, in addition to the codons for the first 96 amino acids of IL-2, contains codons for a bridge link of 6 amino acids and the following codons for hirudin.

The plasmid (51) is reacted with Eco RI and Hind III and the fragment (52) is isolated, this contains the DNA sequence for the aforementioned eukaryotic fusion protein.

The plasmid (2) (Fig. 1) is opened with Eco RI and Hind III. The obtained linearized plasmid (53) is ligated with the DNA sequence (52), whereby the plasmid pK 370 (54) is obtained.

If the plasmid (54) corresponding to example 1 is expressed in E.coli, a fusion protein is obtained in which the bridge link follows the first 96 amino acids of IL-2

and in connection with this amino acid sequence for hirudin.

EXAMPLE 6:

From the plasmid (41) (example 3, fig. 3c), the DNA segment coding for monkey proinsulin is cleaved with the restriction enzymes Eco RI and Hind III, and the ends above are filled. You get the DNA segment (55).

The plasmid (48) (Example 5, Fig. 5) is opened with Sma I and treated with bovine alkaline phosphatase. The obtained linearized plasmid (56) is ligated with the DNA segment (55) whereby the plasmid pK302 (57) is obtained. E.coli 294 cells are transformed with the ligation mixture, whereby clones containing the desired plasmid are first characterized by restriction and then by sequence analysis for plasmid DNA.

From plasmid (57), the segment (58) which codes for IL-2 and monkey proinsulin is cleaved with Eco RI and Hind III.

The plasmid (2) (example 1, fig. 1) is also cleaved with Eco RI and Hind III and the segment (58) is ligated into the linearized plasmid (3). The plasmid pKH 101 (59) is obtained.

Expression according to example 1 leads to a fusion protein, in which the first 96 amino acids for IL-2 are followed by a bridge link of 14 amino acids (corresponding to Y in DNA segment (58)), to which the amino acid sequence for monkey proinsulin joins.

Appendix I; The DNA sequence for interleukin-2

Claims (3)

1. Process for the production of a fusion protein, with a C- or N-terminal portion corresponding to the first 90-132 amino acids of interleukin-2 (IL-2), but not exhibiting any interleukin-2 activity, of the general formula where X is the amino acid sequence for the first 90-132 amino acids of human interleukin-2, Y means a direct link or a bridge link of 2-14 genetically coded amino acids, which enables cleavage of the amino acid sequence Z, and Z is a sequence of genetically coded amino acids, where Y, neighbor to Z, contains Met, Cys, Trp, Arg, Lys, Asp or Pro or consists of these amino acids, characterized by transforming a host cell with a gene structure encoding this fusion protein, culturing the host cell and separating the expressed fusion protein from the the soluble proteins, preferably by centrifugation. 2. Method according to claim 1, characterized in that gene structures are used which code for a fusion protein in which Y, neighbor to Z, contains the amino acid sequence or consists of this. 3. Method according to claim 1 or 2, characterized in that gene structures are used which code for a fusion protein in which Z means the amino acid sequence of a proinsulin or a hirudin.