FI86887B - FUSIONSPROTEIN, FOERFARANDE FOER DERAS FRAMSTAELLNING OCH DERAS ANVAENDNING. - Google Patents

Abstract

1. Claims for the Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE A fusion protein of the general formula Met - Xn - D' - Y - Z in which n is zero or 1, X is a sequence of 1 to 12 genetically codable amino acids, D' is a sequence of about 70 amino acids in the region of the sequence of amino acids 23 - 93 of the D-peptide in the trp operon of E. coli, Y denotes a sequence of one or more genetically codable amino acids which permits the following amino acid sequence Z to be cleaved off, and Z is a sequence of genetically codable amino acids. 1. Claims for the Contracting State : AT A process for the preparation of a fusion protein of the general formula (1) Met - Xn - D' - Y - Z in which n is zero or 1, X is a sequence of 1 to 12 genetically codable amino acids, D' is a sequence of about 70 amino acids in the region of the sequence of amino acids 23 - 93 of the D-peptide in the trp operon of E. coli, Y denotes a sequence of one or more genetically codable amino acids which permits the following amino acid sequence Z to be cleaved off, and Z is a sequence of genetically codable amino acids, characterized by expressing a gene structure which codes for these fusion proteins in a host cell, and separating the fusion protein.

Description

, 86887

Fusion proteins, method of their preparation and their use

The production of smaller eukaryotic proteins with a molecular weight of up to about 15,000 daltons by genetic engineering methods often produces only a small yield in bacteria. The host's own proteases presumably cleave rapidly formed proteins. It is therefore often convenient to prepare such as fusion proteins, especially those containing a host-specific protein moiety to be subsequently removed.

It has now been found that the portion of the D protein corresponding to the E. coli trp operon, consisting of only about 70 amino acids, is particularly well suited for the generation of fusion proteins; this portion is in the range of 23-93 / C of the amino acid sequence. Yanofsky et al .. Nucleic Acids Res. 9 (1981) 66477 and is hereinafter also referred to as "D-peptide". Between the carboxyl terminus of this peptide and the 20 amino acid sequence of the desired eukaryotic protein is a sequence of one or more genetically encodable amino acids that allows chemical or enzymatic cleavage of the desired protein. In preferred embodiments of the present invention, the amino terminus is associated with a short amino acid sequence consisting of Lys-Ala, optionally followed by a sequence of 1-10, especially 1-3 other genetically encodable amino acids, preferably two amino acids, and especially Lys-Gly.

The invention thus relates to a fusion protein of the general formula

Met-X-D1 -Y-Z, n '35 wherein n is 0 or 1; x is a sequence of 1 to 12 amino acids of genetically encodable 86887 2 sa, preferably Lys-Ala; D 'is a sequence of about 70 amino acids in the amino acid sequence of the D-peptide corresponding to the E. coli trp operon; Y is a sequence of one or more 5 genetically encodable amino acids that allows cleavage of the amino acid sequence Z following itself, and Z is a sequence of genetically encodable amino acids.

Other aspects and preferred embodiments Lo of the invention are described below and are defined in the claims.

It is, of course, preferred that the undesirable (host-specific) portion of the fusion protein be as small as possible, since then the cell produces little "ballast" L5 and thus the yield of the desired protein is high. In addition, cleavage of the undesired part results in fewer by-products, which facilitates the handling of the product. The above is counteracted by the fact that the (assumed) hedging effect of the undesired portion is expected only from a certain size of 20. Surprisingly, it has now been found that the D-protein segment selected according to the invention fulfills this function, even though it has only about 70 amino acids.

In several cases, especially in the preferred embodiment where X is Lys-Ala or contains this sequence at its N-terminus, an insoluble fusion protein is formed. This is easily distinguishable from soluble proteins, which greatly facilitates handling and increases yield. The formation of an insoluble fusion protein is surprising, since the bacterial-specific part, about 70 amino acids, is quite small and, on the other hand, is a structural component of the protein dissolved in the host cell.

"About 70 amino acids in the amino acid sequence of the D-peptide in the region 23-93" means that variations are possible in a manner known per se, i.e. 3,86887 can be omitted or individual amino acids can be changed without significantly altering the properties of the fusion proteins of the invention. Such variations are also within the scope of the invention.

Preferably, the desired eukaryotic protein is a biologically active protein, such as hirudin, or a precursor of such a protein, such as human proinsulin.

Preferably, the fusion protein is produced by expression in a suitable system, and in a particularly preferred embodiment, after disruption of the host cells, it is isolated from the precipitate into which it is enriched due to its low solubility. Thus, it is possible to easily separate the cell from the soluble components.

Suitable host cells are mainly bacterial-15 host systems, preferably E. coli, since the bacterial part of the fusion protein is a protein specific for E. coli.

The DNA sequence encoding the fusion protein of the invention is incorporated in a known manner into a vector which is expressed in a well-chosen digestion system.

The promoter and operator used in the bacterial host are preferably the Lac, Tae, PL or PR, hsp, omp or synthetic promoter of the β-phage described, for example, in DE-A-3 430 683 (EP-A-25. 173 149).

Particularly preferred is a vector containing E.

The following elements from the I coli trp operon: promoter, operator, and L-peptide, ribosome binding site. The first 30 amino acids of this L-peptide are particularly conveniently associated with the coding region, followed by amino acids 23-93 of the D-protein after a short amino acid sequence.

:. ·. The intermediate sequence Y, which allows cleavage of the desired polypeptide, is determined by the composition of this desired peptide. For example, when this does not contain methionine, Y may be Met, which may be chemically cleaved with bromocyanine. If the carboxyl terminus of the linker Y is cysteine or Y is Cys, enzymatic cysteine-specific cleavage or chemical cleavage can be performed, for example, after specific S-cyanulation. If the carboxyl terminus of the bridge portion Y is tryptophan or Y is Trp, chemical cleavage with N-bromo-succinimide can be performed. If Y is Asp-Pro, 10 can be done in a manner known per se / 6. Piszkiewics et al. Biochemical and Biophysical Research Communications 40 (1970) 1173-1178 / 7 proteolytic cleavage. The Asp-Pro bond can, as observed, be made even more sensitive with acids when Y is (AspJ ^ -Pro or Glu- (AspJ ^ -Pro, where m is 1, 2 or 3).

In this case, cleavage products are obtained which start at the N-terminus with the Pro group or end at the C-terminus with the Asp group.

Examples of enzymatic cleavages are also known in which modified enzymes with improved specificity / vert C.S. Craik et al., Science 228 (1985) 291-297 /. If the desired eukaryotic enzyme is human proinsulin, the peptide sequence in which the trypsin-cleavable amino acid (Arg, Lys) is bound to the N-terminal amino acid (Phe) of proinsulin, e.g., Ala-Ser-Met-Thr-25 Arg, may be selected as the sequence Y, for perform arginine-specific cleavage with trypsin protease. If the desired protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, cleavage by factor Xa is also possible (EP-A-0 161 937).

In the formation of section Y, it is also possible to take advantage of synthetic possibilities and to construct suitable restriction enzyme cleavage sites. Thus, the DNA sequence corresponding to the amino acid sequence Y can also act as a linker or adapter sequence.

Expression as a fusion protein 35 according to the invention preferably takes place under the control of the E. coli trp operon, a DNA sequence containing the promoter and operator of the trp-86887 operon is commercially available, expression as proteins under the control of the trp operon has been extensively described in the literature, for example in EP Application Publication No. 5,0 036 776 (A2). induction of the trp operon can be achieved in the absence of L-tryptophan from the medium and / or in the presence of indolyl-3-acrylic acid in the medium.

Under the control of the trp operon, transcription of the 14-amino acid L-peptide first occurs.

This L-peptide contains L-tryptophan at positions 10 and 11, respectively. The rate of L-peptide protein synthesis determines whether the next structural gene is translated at the same time or whether protein synthesis is disrupted. When L-tryptophan is deficient, slow synthesis of L-peptide occurs due to the low concentration of tRNA corresponding to L-tryptophan, followed by synthesis of the next protein. In contrast, when the L-tryptophan content is high, the corresponding mRNA sequence is read rapidly, and protein biosynthesis is disrupted because the mRNA has a terminator-like structure / C. Yanofsky et al., Loc. Sit ^.

The translation density of the mRNA is strongly influenced by the quality of the nucleotides close to the start codon, therefore, in the case of fp protein expression by the trp operon, it is preferable to use nucleotides corresponding to the first 25 amino acids of the L-peptide as the beginning of the fusion protein structural gene. In a preferred embodiment of the invention, in connection with the trp system, the nucleotides corresponding to the first three amino acids of the L-peptide (hereinafter the L'-peptide) were selected as codons corresponding to the N-terminal amino acids of the fusion protein.

The invention therefore also relates to expression vectors for fusion proteins, preferably plasmids, in which the DNA of the vector contains, from the 5 'end, the following characteristic parts (in the appropriate order and phase): the pro-35 engine, the operator, the ribosome binding site and the structural gene of the fusion protein. contains the amino acid sequence I (Appendix) before the sequence of the desired protein. In front of or as the first triplet of the structural gene is an start codon (ATG) and possibly additional codons corresponding to other amino acids located between the start codon and the D 'sequence or D' sequence and the gene corresponding to the desired protein. Depending on the amino acid composition of the desired protein, a DNA sequence preceding the structural gene is selected that allows pg of the desired protein to be cleaved from the fusion protein.

In the context of expression as a fusion protein of the invention, it may be appropriate to modify individual triplets corresponding to the first amino acids after the ATC start codon to prevent possible base pairing at the 15 mRNA level. Such alterations, as well as alterations, deletions, and additions to individual amino acids of the D 'protein, are known to those skilled in the art and are also within the scope of the invention.

Because of the many advantages of the smaller plasmids, in a preferred embodiment of the invention, the DNA sequence containing the tBR322-based tetracycline resistance gene is deleted from the plasmids. Preferably, a segment extending from the HindIII cleavage site at position 29 to the PvuII cleavage site at position 2066 is removed. In the case of the plasmids according to the invention, it is particularly expedient to delete an even slightly larger DNA sequence, taking advantage of the PvuII cleavage site at the beginning (reading direction) of the trp operon (which is located in the non-essential part). Thus, the resulting large fragment with a PvuII cleavage site at both ends can be ligated directly. The resulting plasmid, truncated by about 2 keP (kilobase pairs), results in enhanced expression, possibly leading to an increase in copy number in the host cell.

7 86887

The invention is further illustrated by the following examples.

Example 1 a) Chromosomal E. coli DNA is digested with Hinfl, 5 and a 492 bp fragment containing the codons corresponding to the first six amino acids of the promoter, operator, L-peptide structural gene, attenuator and trp-E structural gene is isolated from the trp operon. . This fragment is supplemented with Klenow polymerase with deoxynucleotide-10 triphosphates, ligated at both ends into an oligonucleotide containing a HindIII recognition site, and digested again with HindIII. The HindIII fragment thus obtained is ligated to the HindIII cleavage site of pBR322. Thus, plasmid ptrpE2-1 / J.C. is obtained. Edmann et al., Nature 291 (1981) 15503-506.7 · This is converted to plasmid ptrpL1 as described in the literature.

Synthetic oligonucleotides (N1) and N2) 5 'CGA CAA TGA AAG CAA AGG 3' (N1) 5 'CCT TTG CTT TCA TTG T 3' (N2) 20 complementary to double-stranded oligo-nucleotide (N3) 5 'CGA CAA TGA AAG CAA AGG 3 '/ kto.

(N3) 3 'T GTT ACT TTC GTT TCC 5' generates a DNA sequence corresponding to the first three amino acids of the L-peptide at the ClaI site of plasmid ptrpL1 as well as a restriction site (Stul) for insertion of other DNA (Figure 1). Plasmid ptrpL1 is reacted with the enzyme ClaI according to the manufacturer's instructions, the mixture is extracted with phenol, and the DNA is precipitated with ethanol. The opened plasmid is reacted with alkaline phosphatase to remove phosphate groups at the 5 'ends. The synthetic nucleotides are phosphorylated at their 5 'ends and ligated into a phosphatase-treated, opened plasmid using T4 DNA ligase. Upon completion of the ligation reaction, transformation of E. coli 35,294 and selection of transformants based on the presence of Amp-86887 8 resistance and the Stul restriction site follow.

Approximately 80% of the clones obtained had the expected restriction site; the nucleotide sequence given in Figure 1 was determined by sequence analysis. Plasmid pH120 / 14 is obtained, which contains the nucleotide triplets (L'-peptide) corresponding to the first three amino acids of the L-peptide after the ribosome binding site of the L-peptide and then the Stul site, which in turn allows the insertion of additional DNA and thus formation of fusion proteins with the first three to 10 amino acids of the L-peptide.

b) The following is illustrated using the above oligonucleotide (N1) as an example for the chemical synthesis of such an oligonucleotide: M.J. Gaitin et al. Nucleic Acids 15 Research 8 (1980) 1081-1096) covalently binds a nucleoside at the 3 'end, in this case guanosine, to a glass bead support / CPG (= controlled pore glass) LCAA (= long chain alkylamine). ni, long chain alkyl amine), manufactured by the 20 hydroxyl functional group at the Pierce / 3 'end. Guanosine is then reacted as an N-2-isobutyroyl-3'-O-succinoyl-5'-dimethoxytrityl ether with a modified carrier in the presence of N, N1-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to give the succinoyl group a free carboxyl group. the amino group of the long chain amine on the support.

In the following synthetic steps, the base components in the form of 51-O-dimethoxytrityl nucleoside-3'-phosphoric acid monomethyl ester dialkylamide or chloride are used, wherein the adenine is N6-benzoyl compound

40 mg of the carrier bound with 1 .mu.mol of guanosine are treated successively with the following substances: 35 a) methylene chloride, b) 10% trichloroacetic acid in methylene chloride, 9 86887 c) methanol, d) tetrahydrofuran, e) acetonitrile, f) 15 .mu.mol the corresponding nucleoside phosphite and 70 μmol of 5 tetrazole in 0.3 ml of anhydrous acetonitrile (5 min), g) 20% acetic anhydride in tetrahydrofuran containing 40% lutidine and 10% dimethylaminopyridine (2 min), h) tetrahydrofuran, 10) tetrahydrofuran containing 20% water and 40% lutidine, j) 3% iodine in a mixture containing collidine, water and tetrahydrofuran in a volume ratio of 5: 4: 1, k) tetrahydrofuran and 15 l) methanol.

As used herein, "phosphite" means a deoxyribose-3'-monophosphoric acid monomethyl ester in which the third valence is saturated with chlorine or a tertiary amino group, for example a diisopropylamine group.

The yield of the individual synthetic steps can in each case be determined spectrophotometrically after the detritylation reaction b) by measuring the absorbance of the dimethoxytrityl cation at 496 nm.

When oligonucleotide synthesis is complete, the methyl phosphate protecting groups of the oligomer are removed with p-thiocresol and triethylamine.

The oligonucleotide is then removed from the solid support by treatment with ammonia for 3 hours. Treatment of the oligomers with concentrated ammo-30 for 2-3 days quantitatively removes the amino protecting groups of the bases. The crude product thus obtained is purified by high pressure chromatography (HPLC) or polyacrylamide gel electrophoresis.

Other oligonucleotides are synthesized in a completely similar manner.

86887 10 c) Plasmid ptrpE5-1 /R.A. Hallewell et al.f Gene 9 (1980) 27-477 is reacted with the restriction enzymes HindIII and SalI according to the manufacturer's instructions, and a DNA-5 fragment of about 620 base pairs is removed. Synthetic oligonucleotides (N4) and (N5) 5 'AGC TTC CAT GAC GCG T 3' (N4) 5 'ACG CGT CAT GGA 3' (N5) are phosphorylated, co-incubated at 37 ° C and ligated with DNA ligase into the corresponding proinsulin. to blunt-ended 10 DNA sequences / W. Wetwkam et al., Gene 19 (1982) 179-1837. Following the reactions with HindIII and SalI, the extended proinsulin DNA is thus covalently ligated with the enzyme T4 DNA ligase into the plasmid opened to give plasmid pH106 / 4 (Figure 2).

Plasmid pH106 / 4 is then reacted once more with SalI, supplemented with single-stranded ends with Klenow polymerase to blunt ends, and then incubated with the enzyme MstI. A DNA fragment of about 500 base pairs containing the entire proinsulin-20 coding portion and a sequence encoding about 210 base pairs of the D protein from the E. coli trp operon are isolated. This DNA fragment is blunt-ended and is inserted into the Stul site of plasmid pH12Q / 14 to give plasmid pH154 / 25 (Figure 3). This is applicable to fusion protein expression under the control of the trp operon, in which the L'- and D1-peptide is followed by the amino acid sequence Ala-Ser-Met-Thr-Arg, which is associated with the amino acid sequence of proinsulin.

Example 2 Plasmid pH154 / 25 (Figure 3) is reacted with the restriction enzymes BamHI and Xmall1. Single-stranded ends are supplemented with Klenow polymerase and ligated using T4 DNA ligase. Plasmid pH254 (Figure 4) is obtained, which is suitable for the production of a fusion protein containing the amino acid sequence L ', D'-proinsulin 35 under the control of the trp promoter. The plasmid is somewhat lower than pH 154/25, which may be advantageous.

Example 3

By incubating plasmid pH254 (Example 2, Figure 4) with the restriction enzymes MluI and SalI, a 280 bp DNA sequence is excised and deleted. The remainder of the plasmid is blunt-ended with Klenow polymerase and covalently cyclized back using DNA ligase. Thus, plasmid pH255 (Figure 4) is obtained, in which the structural gene can be inserted at one of the restriction sites MluI,

Sali and EcoRI. Under induction conditions, a fusion protein containing an L * -D 'protein sequence is formed. Naturally, other restriction sites can be added to plasmid pH255 using suitable linkers.

15 Example 4

Plasmid pH154 / 25 (Figure 3) is incubated with the enzymes MluI and EcoRI, and the released DNA fragment (approximately 3Q0 base pairs) is removed. The remainder of the plasmid is supplemented with Klenow polymerase. Ring closure is performed by DNA ligase 20. A structural gene can be inserted into the EcoRI site of the resulting plasmid pH256 (Figure 5).

Example 5

Deletion of the 600 bp fragment from plasmid pH256 (Example 4, Figure 5) with the restriction enzymes BamHI and 25 NruI gives plasmid pH257 (Figure 5). This is done by first incubating pH256 with BamHI and forming blunt ends with Klenow polymerase. After incubation of the plasmid with NruI and removal of the 600 bp fragment, pH257 is established by incubation with DNA ligase.

30 Example 6. . By locating the Lac repressor /P.J. Farabaugh, Natu re 274 (1978) 765-7697 to plasmid pKK 177-3 / Amann et al., Gene 25 (1983) 1677 gives plasmid pJF118. This is reacted with EcoRI and SalI, and the remaining 35 parts of the plasmid are isolated.

12 8 6 8 S 7

A fragment of approximately 495 base pairs is separated from plasmid pH106 / 4 (Figure 2) by SalI and incubation with Mstl.

Synthetically prepared oligonucleotides (N6) 5 and (N7) 5 'ACG AAT TCA TGA AAG CAA AGG 3' (N6) 5 'CCT TTG CTT TCA TGA ATT CGT 3' (N7) are phosphorylated and ligated to the blunt-ended DNA fragment by DNA ligase . By reacting with EcoRI and SalI 10, complementary ends are released that allow ligation to the opened plasmid pJFH8.

After transformation of E. coli with the hybrid plasmid thus obtained, the correct clones are selected on the basis of the size of the restriction fragments. This plasmid is designated 15 pJ120 (Figure 6).

Production of fusion proteins is performed in shake flasks as follows. E. coli transformants containing plasmid pJ120 have been cultured overnight in LB medium (JH Miller, Experiments on Molecular Genetics, Cold Spring 20 Harbor Laboratory, 1972) supplemented with 50 μg. / ml ampicillin, dilute the culture at approximately li 100, and monitor growth by measuring optical density.

When the optical density (OD) is 0.5, isopropyl-D-galactopyranoside (IPTG) is added to a concentration of 1 mM, and the bacteria are separated after 150-180 minutes by centrifugation.

The bacteria are boiled for 5 min in a buffer mixture (7 mol / l urea, 0.1% SDS, 0.1 mol / l sodium phosphate, pH 7.0), and the samples are transferred to SDS gel electrophoresis plates. Electrophoresis yields a protein band from bacteria containing plasmid pJ120 that is the size of the expected fusion protein and reacts with insulin antibodies. Once the fusion protein is isolated, the desired pro-insulin derivative can be released by bromocyanine cleavage. After the bacteria are disrupted (French press; Dyno-Miihle) and centrifuged, the L1-D'-proinsulin fusion protein is in the precipitate, 13 86887, so that considerable amounts of other proteins can already be separated into the supernatant.

The induction conditions given apply to shake cultures; in the case of larger fermentations, correspondingly modified OD values and possibly slightly modified IPTG concentrations are appropriate.

Example 7 E. coli transformants containing plasmid pH154 / 25 (Figure 3) are cultured overnight in LB medium containing 5 μg / ml ampicillin and the culture is diluted the following morning at approximately 1: 100 in M9 medium (JH Miller , loc.cit.) containing 2000 ug / ml casamino acids and 1 ug / ml thiamine. With an OD of 0.5, indolyl-3-acrylic acid is added to a final concentration of 15 μg / ml. After 2-3 15 hours of incubation, the bacteria are separated by centrifugation. SDS electrophoresis shows, at the expected site for the fusion protein, a fairly distinct protein band that reacts with insulin antibodies. After disruption of the bacteria and centrifugation, the L ', D'-proinsulin fusion protein-20 is precipitated, so that again considerable amounts of other proteins remain in the supernatant.

Also in this case, the given induction conditions for shake cultures apply. Larger volumes of fermentation require altered casaminic acid concentrations or the addition of L-tryptophan.

Example 8

Plasmid pH154 / 25 (Figure 3) is opened with EcoRI, and the single-stranded ends are supplemented with Klenow polymerase. The DNA thus obtained is incubated with the enzyme MluI, and the DNA encoding the insulin is detached from the plasmid. By performing the electrophoretic treatment, this fragment is separated from the remainder of the plasmid, and this remainder is isolated.

The plasmid shown in Figure 3 of DE-A-3 249 430 / EP-A-0 171 024.7 is reacted with the restriction enzymes Accl and SalI, and a DNA fragment containing the hiru-86887 14 din sequence is isolated. After the single-stranded ends of the SalI cleavage sites are supplemented with Klenow polymerase, the DNA segment is ligated to synthetic DNA of formula (N8).

5 Met Thr 5 'CCC ACG CGT ATG ACG T 3' „.Ttn (No) 3 'GGG TGC GCA TAC TGC ATA 5'

The ligation product is incubated with MluI. After inactivation of the enzyme at 65 ° C, the DNA mixture is treated with alkaline bovine phosphatase for one hour at 37 ° C. The phosphatase and restriction enzyme are then removed from the mixture by phenol extraction, and the DNA is purified by ethanol precipitation. The DNA thus treated is placed in the opened remainder of plasmid pH154 / 25 with T4 ligase to give plasmid pK150, which was characterized by restriction analysis and Maxam-Gilbert DNA sequencing (Figure 7).

Example 9 E. coli 294 bacteria containing plasmid 20 pK15Q (Figure 7) are grown in LB medium containing? Q-5Qy.ug/ml ampicillin overnight at 37 ° C. The culture is diluted 1: 100 in M9 medium containing 20000 ug / tnl of cascinoic acids 1 ug / ml of amine and incubated at 37 ° C with constant agitation. When the OD600 is 0.5 or 25, indolyl-3-acrylic acid is added to a final concentration of 15 [mu] g / m] and incubated for 2-3 or 16 hours. The bacteria are then separated by centrifugation and disrupted in 0.1 M sodium phosphate buffer (pH 6.5) under pressure. Sparingly soluble proteins are separated by centrifugation, and analyzed by SDS-polyacrylamide electrophoresis. It turns out that cells with an induced trp operon contain a novel protein of less than 20,000 but greater than 14,000 daltons that was not present in the uninduced cells. Isolating the fusion protein and reacting it with bromocyanine releases hirudin.

15 8 68 57

Example 10

The following describes the structures that allow the placement of DNA sequences in front of the 5 · end of the trp-D sequence with as many different restriction enzyme recognition sites as possible in order to place the trp-D sequence as widely as possible in a wide variety of prokaryotic expression systems.

Plasmids pUC12 and pUC13 (Pharmacia PL Biohemicals, 54Q1 St. Goar: The Molecular Biology Catalog 1983, Appendix IQ s 89) contain a poly-linker sequence whereby plasmid pUC13 can be inserted between restriction sites XmaI and SacI from plasmid p106 / 4 (Figure 2). the resulting MstI-Hind-III trp fragment fused to the HindIII-hirudin-SacI fragment from plasmid pK150 (Figure 7). This is done by treating the DNA of plasmid pUC13 with the restriction enzyme XmaI. The ends of the linearized plasmid are supplemented with Klenow polymerase. After precipitation with ethanol, the DNA is treated with SacI and reprecipitated from the reaction mixture with ethanol.

The DNA is then reacted in an aqueous ligation mixture with the Mst-HindIII-trp-D fragment isolated from plasmid pH106 / 4 and the HindIII-SacI hirudin fragment isolated from plasmid pK150 in the presence of T4 DNA ligase.

Plasmid pK160 thus obtained now contains, immediately before the start of the trp-D sequence, a multiple restriction enzyme recognition sequence with cleavage sites for the enzymes XmaI, SmaI, BamHI, XbaI, Hindi, SalI, Accl,

PstI and HindIII. This construct further provides an EcoRI cleavage-30 site 3 'to the 3' end of the hirudin sequence (Figure 8).

Example 11

Plasmid pH131 / 5 is prepared as follows according to Figure 1 of Example 1 of DE patent application 35 14 113): Plasmid ptrpL1E). Edman et al., Nature 291 (1981) 35 503-5067 is opened with Clalr and ligated synthetically to 86887 16 prepared self-complementary oligonucleotide (N9) 5 'pCGACCATGGT 3' (N9)

The plasmid pH131 / 5 thus obtained (Fig. 9) is opened from the NcoI restriction site formed, and the formed single-stranded ends are supplemented with Klenow polymerase. The linearized homogeneous DNA is then digested with EcoRI, and the larger of the resulting DNA sequences is separated from the smaller one by precipitation with ethanol. The residual DNA of plasmid pH131 / 5 thus obtained is then ligated to the trp-D'-hirudin-encoding fragment from pK160 by T4 ligase. This fragment was isolated from plasmid pK160 by digestion with Hindi and EcoRI. The fragment is separated from the remaining 25 parts of the plasmid by gel electrophoresis and then eluted from the gel. The ligation product transforms E. coli K12. Clones containing plasmid DNA are isolated and characterized by restriction analysis and DNA sequencing. Plasmid pK170 thus obtained, fused to the trp operon, contains a DNA sequence encoding the sequence Met-Asp-Ser-Arg-Gly-Ser-Pro-Gly-trp-D '- (hirudin) (Figure 9).

Example 12

Plasmid pJF118 (Example 6) is opened with EcoRI, and the resulting single-stranded DNA ends are blunt-ended with Klenow polymerase. DNA thus treated

then digested with SalI and gel electrophoretically separated from the short EcoRI-SalI fragment.

Plasmid pK170 (Example 11) is digested with NcoI, and the single-stranded ends are complemented with Klenow polymerase 30. Plasmid DNA is separated from the reaction mixture by ethanol precipitation and treated with HindIII and BamHI. Two of the resulting fragments are isolated, namely the NcoI (ends amplified) -trpD'-HindIII fragment and the HindIII-hirudin-BamHI fragment (DE-A-35 3 429 430). Both fragments are isolated by gel electrophoresis.

86887 17

In addition, the BamHI-SalI-hirudin-1 fragment according to Figure 2 of DE-A-3 429 430 is isolated. The four fragments, i.e., the remainder of plasmid pJF118, the NcoI-trpD'-HindIII fragment, the HindIII-hiru-5 din-BamHI fragment, and the hirudin-11 fragment, are then ligated together and transformed with the resulting plasmid pK180 (Figure 10). E. coli K12-W 3110. The correct plasmids are identified by the presence of an EcoRI-trpD'-hirudin-SalI fragment in the plasmid DNA. The trpD'-hirudin sequence 10 is now linked to the tac promoter. The fusion protein is produced according to Example 6.

For example 13

In plasmids derived from pBR322, such as pH120 / 14 (Example 1, Figure 1), pH154 / 25 (Example 15 1, Figure 3), pH256 (Example 4, Figure 5), pK150: (Example 8, Figure 7) and pK 170 (Example 11, Figure 9) have - in clockwise directions - between the start codon of the fusion protein and the next HindIII site (corresponding to the HindIII site at position 29 of pBR322). PvuII site in the region of the fragment containing the trp promoter and operator, but outside the promoter region.

It has now been found that deleting the DNA sequence flanked by the PvuII site described above and the PvuII site corresponding to position 2066 in pBR322 clearly increases the yield of the cloned protein (i.e., fusion protein).

The following describes by way of example the truncation of plasmid pH154 / 25 to plasmid pH154 / 25x, which can be done in a similar way for the other plasmids mentioned above (in the same way as the abbreviated plasmids are denoted by an asterisk): pH154 / 25 is reacted (according to the manufacturer's instructions) to form three fragments: 35 Fragment 1:

From the PvuII restriction site of proinsulin to the PvuII restriction site of 86B 872 of position 868 87 18 of pBR322,

Fragment 2: from the PvuII restriction site near the trp promoter to the PvuII site of proinsulin and Fragment 3: from the PvuII site near the trp promoter fragment to the PvuII site corresponding to position 2066 of pBR322.

Fragments can be separated by agarose electrophoretically and then isolated (Maniatis et al., Molecular Cloning Cold Spring Harbor 1982). Fragments 1 and 2 are ligated together under "blunt end" conditions using the enzyme T4 DNA ligase. After transformation of E. coli 294, it is determined which columns contain the plasmid containing the complete proinsulin sequence and the fragments in the desired order. Figure 11 shows plasmid pH154 / 25x.

When making the expression as described in the above examples, a clear increase in the proportion of fusion protein is observed.

Example 14 Plasmid pH154 / 25x (Example 13, Figure 11) is treated with HindIII and SalI, and a small fragment (containing the proinsulin sequence) is separated by gel electrophoresis. The large fragment is isolated and ligated to synthetic DNA (N10).

25 (Lower) Trp Glu Asp Pro Met Ile Glu (Gly) (Arg) A GCT TGG GAG GAT CCT ATG ATC GAG GG (N10) ACC CTC CTA GGA TAC TAG CTC CCA GCT Plasmid plntl3 is obtained (Figure 13).

DNA (N10) encodes an amino acid sequence containing several cleavage sites suitable for chemical cleavage: a) Met for bromocyanine, b) Trp for N-bromosuccinimide (NBS or BSI) c) Asp-Pro for proteolytic cleavage, with Glu above 35 still the sensitivity of the Asp-Pro bond to the action of acids.

Thus, this insertion of the HindIII-SalI linker (N10) into the reading frame of the encoded polypeptide provides the aforementioned ability to perform chemical cleavage, depending on the amino acid sequence of the desired protein or its cleavage reagent-5 sensitivity.

Images are not to scale.

20 86887

Aminohapposekvenssi_I

2 3

Ser Asn Orly His Asn Vai Val lie l o

Tyr Arg Asn His lie Pro Ala Gin 20

Thr Leu lie Glu Arg Leu Ala Thr 3 o

Met Ser Asn Pro Val Leu Met Leu 4 o

Ser Pro Gly Pro Gly Val Pro Ser

Glu Ala Gly Cys Met Pro Glu Leu 5 o

Leu Thr Arg Leu Arg Gly Lys Leu 6 0

Pro lie lie Gly lie Cys Leu Gly / o ^ 9 3

His Gin Ala He Val Glu Ala 21 868 87

DNA sequence I

Ser Asn Gly His Asn Vai Vai Ile

AGC AAT GGG CAT AAC GTG GTG ATT

. ·

1 O

Tyr Arg Asn His Ile Pro Ala Gin

TAC CGC AAC CAT ATA CCG GCG CAA

• · 20

Thr Leu Ile Glu Arg Leu Ala Thr

ACC TTA ATT GAA CGC TTG GCG ACC

5 0 # ♦ • 30

Met Ser Asn Pro Vai Leu Met Leu

ATG AGT AAT CCG GTG CTG ATG CTT

• # 40

Ser Pro Gly Pro Gly Or Pro Ser

TCT CCT GGC CCC GGT GTG CCG AGC

100 *

Glu Ala Gly Cys Met Pro Glu Leu

GAA GCC GGT TGT ATG CCG GAA CTC

• · 50

Leu Thr Arg Leu Arg Gly Lys Leu

CTC ACC CGC TTG CGT GGC AAG CTG

150 and 60

Pro Ile Ile Gly Ile Cys Leu Gly

CCC ATT ATT GGC ATT TGC CTC GGA

• »· 70

His Gin Ala Ile Vai Glu Ala

Cat CAG GCG ATT GTC GAA GCT

20 0

Claims (12)

22 868S7 A fusion protein, characterized in that it has the general formula Met-Xn-D'-Y-Z, in which n is zero or 1; X is a sequence of 1 to 12 genetically assembled amino acids; D '10 is a sequence of about 70 amino acids in the amino acid sequence of the D-peptide corresponding to the E. coli trp operon; Y is a sequence of one or more genetically encodable amino acids that allows cleavage of the amino acid sequence Z 15 following itself, and Z is a sequence of genetically encodable amino acids. Fusion protein according to Claim 1, characterized in that n is 1 and X comprises at most 5 amino acids, in which case X is preferably 20 N-terminal sequence Lys-Ala. Fusion protein according to Claim 1 or 2, characterized in that Y contains a C-terminal group Met, Cys, Trp, Arg, Lys or one of the groups (Asp? -Pro, Glu- (Asp) m-Pro or Ile-). Glu-Gly-Arg, wherein m is 1, 2 or 3, or consists of these amino acids or one of these groups. Fusion protein according to one or more of the preceding claims, characterized in that Z is the amino acid sequence of human proinsulin or hirudin. A method for producing a fusion protein according to one or more of claims 1 to 4, characterized in that the gene structure encoding this fusion protein is expressed in a bacterial host cell, preferably E. coli, and the fusion protein is isolated. 23 8 6 8 8 7 A method according to claim 5, characterized in that in the gene construct the sequence (Dr) encodes DNA sequence I (Annex) and / or in the gene construct encodes the sequence X DNA sequence (coding strand) 5 5 'AAA GCA AAG GGC 3f, and / or the gene construct is selected such that the fusion protein is insoluble and / or the gene construct is in phase in a vector containing a promoter, operator and ribosome binding site from the E. coli trp operon. The method of claim 6, wherein the vector is a pBR322 derivative in which a sequence extending from the HindIII site at position 29 to the PvuII site at position 2066 has been removed from the pBR322 DNA. Gene construct, characterized in that it encodes fusion proteins according to Claims 1 to 4. A vector, preferably a derivative of plasmid pBR322, in which a sequence extending from the HindiII position at position 29 to the PvuII position at position 2066 has been deleted from the pBR322 DNA, characterized in that it contains the gene construct according to claim 8. Bacterial host cell expression system, preferably an E. coli system, characterized in that it contains a vector according to claim 9. A method for producing a eukaryotic protein, characterized in that the amino acid sequence Z is cleaved chemically or enzymatically from the fusion protein according to claims 1 to 4. Plasmid, characterized in that it is pH154 / 25 (Figure 3), pH254 (Figure 4), pH255 (Figure 4), pH256 (Figure 5), pH257 (Figure 5), pH120 / 14 (Figure 1), pK150 (Figure 7), pK160 (Figure 8), pK170 (Figure 9), pK180 (Figure 10), pH154 / 25 * (Figure 11), pH256 *, corresponding to the pH256 shown in Figure 5, of which PvuII: 11a the fragment obtained has been removed as shown in Figure 11, pH120 / 14 *, which corresponds to pH120 / 14 shown in Figure 1, from which the fragment obtained with PvuII has been removed as shown in Figure 11, pK150 *, which corresponds to Fig. 7 shows pK150 from which the PvuII-derived fragment has been removed as shown in Fig. 11, pK170 * corresponding to pK170 shown in Fig. 9, from which the PvuII-derived fragment has been removed as shown in Fig. 11, or plnt13 (Fig. 12). 25 8 6 8 S 7