CA1336329C - Fusion proteins, a process for their preparation and their use - Google Patents
Fusion proteins, a process for their preparation and their useInfo
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- CA1336329C CA1336329C CA000514682A CA514682A CA1336329C CA 1336329 C CA1336329 C CA 1336329C CA 000514682 A CA000514682 A CA 000514682A CA 514682 A CA514682 A CA 514682A CA 1336329 C CA1336329 C CA 1336329C
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- sequence
- fusion protein
- amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Abstract
A segment of about 70 amino acids from the D-protein of the trp operon of E. coli is suitable for the construction, by genetic engineering methods, of fusion proteins which can contain, upstream of the N-terminal end of the desired protein, a short amino acid sequence of genetically codable amino acids, which is preferably Lys-Ala or contains Lys-Ala at the N-terminal end.
Description
I 3363~
Fusion proteins, a process for their preparation and their use In the preparation by genetic engineering methods of rela-tively small eukaryotic proteins with a molecul~r ~eight up to about 15000 Daltons the yield obtained in bacteria is frequently only small. It is presumed that the proteins which are formed are rapidly degraded by proteases intrin-sic to the host. For this reason, proteins of this type - are advantageously prepared as fusion proteins, in particu-Lar having a portion of protein intrinsic to the host, which is then cleaved off.
It has now been found that a segment composed of only about 70 amino acids of the D-protein from the trp operon of E.
coli is particularly suitable for the formation of fusion proteins, specifically in the region of the sequence of amino acids 23 to 93 (C. Yanofsky et al., NucLeic Acids Res.
9 (1981)6647), also caLled "D'-peptide" hereinafter. Be-tween the carboxyl terminal end of this peptide and of the amino acid sequence of the desired eukaryotic protein there is a sequence comprising one or more genetically codabLe Dmino acids which permits the desired protein to be cleaved off chemicalLy or enzymatically. In preferred em-bodiments of this invention, the amino terminal end ;s foL-lowed by a short amino acid sequence composed of Lys-Ala, optionally followed by a sequence of 1 to 10, in particular 1 to 3, other genetically codable amino acids, preferably by two amino acids, in particular by Lys-Gly.
Hence the invention relates to a fusion protein of the general formula Met-Xn-D'-Y-Z
in which n is zero or 1, i, ~ - 2 - l 33~329 X is a sequence of 1 to 12 gen~t~cally codable amino aC~s, preferably Lys-Ala, D' is a sequence of about 70 amino acids in the region o~f 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 representing the desired final protein.
The present invention will now be described in association with preferred embodiments with reference to accompanying drawings, in which:
Figure 1 shows a process for preparing plasmid pH120/14, which is suitable ~or the expression of a fusion protein having the first three amino acids of the L-peptide.
Figure 2 shows a process for preparing plasmid pH106/4 coding for proinsulin.
Figure 3 shows a process for preparing plasmid pH154/25, which is suitable for the expression of a fusion protein under the control of the trp operon, in which the amino acid sequence Ala-Ser-Met-Thr-Arg is located after the L'- and D'-peptide and is followed by the amino acid sequence of proinsulin.
Figure 4 shows a process for preparing plasmid pH254, which is suitable for the expression of a fusion protein having the amino acid sequence L', D~-proinsulin under the control of the trp promoter, and a process for preparing plasmid pH255 which is suitable for the insertion of a structural gene into one of the restriction sites MluI, SalI and EcoRI.
Figure 5 shows a process for preparing plasmid pH256 which is suitable for the insertion of structural genes into the EcoRI
site, and a process for preparing plasmid pH257.
1~
~ - 2a .,.
Figure 6 shows a process for ~ ~ ing plasmid pJ1~ -coding for proinsulin.
Figure 7 shows a process for preparing plasmid pK150 coding for hirudin.
Figure 8 shows a process for preparing plasmid pK160, which contains, immediately upstream of the trp-D sequence, a multiple restriction enzyme recognition sequence which embraces restriction sites for the enzymes XmaI, SmaI, BamHI, XbaI, HincII, SalI, AccI, PstI and HindIII, and an EcoRI restriction site downstream of the 3'-end of the hirudin sequence.
Figure 9 shows a process for preparing plasmid pK170 which contains a DNA sequence which codes for Met-Asp-Ser-Arg-Gly-Ser-Pro-Gly-trp-D'-(hirudin) fused onto the trp operator.
Figure 10 shows a process for preparing pK180 coding for hirudin.
Figure 11 shows a process for preparing pH154/25 * coding for proinsulin.
Figure 12 shows a process for preparing plasmid pint 13 coding for an amino ac`id sequence allowing chemical cleavage by cyanogen bromide, n-bromosuccinimide or acids.
~ ~ - 2b ' .. , , ;
Further aspects of the invention and preferred embodiments are described here;nafter and defined in the patent claims.
Of course, it i~ advantageous if the undesired portion (in-trinsic to the host) of the fusion protein is as small as possible since then the cell produces only little "ballast"
and hence the yield of desired protein is high. Further-more, when the undesired portion is cleaved off, fewer by-products are produced, which fac;litates working up~ A
factor opposing this is that the (assumed) "protective function" of the undesired portion is to be expected only above a certain size. It has now emerged, surprisingly, that the segment chosen according to the ;nvention from the D-protein fulfils this task although it contains only about 70 amino acids.
In many cases, especially in the preferred embodiment in which X represents Lys-Ala or contains this sequence at the ~-terminal end, the fusion protein formed is insoluble.
The latter can easiLy be separated from the solubLe pro-teins, which great~y facilitates the working up and in-creases the y;eld. The formation of an insolubLe fusion prote;n is surpr;s;ng since, on the one hand, the bacterial portion of only about 70 amino acids ;s quite small and, on the other hand, it is a constituent of a protein which is present in solution in the host cell~
"About 70 amino ac;ds in the region of the sequence of amino _ 3 _ 1336329 acids 23 to 93 of the D-peptide" means that it is possible to carry out, in a manner known per se, variations, that is to say it is poss;ble for individual amino acids to be deleted, replaced or exchanged without this significantly changing the properties of the fusion proteins according to the invention. The invention likewise relates to vari-ations of this type.
The desired eukaryotic protein is preferably a biologicalLy active protein, such as a hirudin, or a precursor of a pro-tein of this type, such as human proinsulin.
The fusion protein is obtained by expression in a suitablesystem, and in the particularly preferred embodiment is, after disruption of the host cells, isolated from the sedi-ment in which it is concentrated owing to its sparing solu-bility. Hence it is easy to separate it from the solubLeconstituents of the cell.
Suitable host cells are all those for which expression sys-tems are known , that is to say mammalian cells and micro-organisms, preferably bacteria, in particular E. coli since, after all, the bacterial portion of the fusion protein is a protein intrinsic to the host E. coli.
The DNA sequence which codes for the fusion protein accord-ing to the invention is incorporated, in a known manner, into a vector which ensures satisfactory expression in the selected expression system.
In bacterial hosts, it is expedient to choose the promoter and operator from the group comprising Lac, Tac, PL or PR of the phage ~, hsp, omp or a synthetic promoter, such as are described, for example, in German Offenlegungs-schrift 3,430,683 (European Patent Application 0,173,149)~
A particularly suitable vector is one which contains the following elements of the trp operon of E. coli: the pro-4moter, the operator and the ribosome binding site of the L-peptide. It is particularly advantageous for the first three amino acids of this L-peptide to follow in the cod-ing region, and then to be followed by a short amino acid sequence and the amino acids 23 to 93 of the D-protein in the trp operon.
The intermediate sequence Y which makes it possible to cleave off the desired polypeptide depends on the compo-sition of this desired peptide: for example, if this con-tains no methionine it is possible for Y to denote Metand then chemical cleavage with cyanogen bromide is car-ried out. If there is cysteine at the carboxyl terminal end in the connecting member Y, or if Y represents Cys, then cysteine-specific enzymatic cleavage or chemical cleav-age, for example after specific S-cyanylation, can be car-ried out. If there is tryptophan at the carboxyl terminal end of the bridging member r, or if Y represents Trp, then chemical cleavage with N-bromosuccinimide can be carried out. If Y represents Asp-Pro, then proteolytic cleavage can be carried out in a manner known per se (D. Piszkie~icz et al., Biochemical and Biophysical Research Communications 40 (1970) 1173-1178). The Asp-Pro bond can, as has been found, be made even more acid-labile if Y is (Asp)m-Pro or Glu-(Asp)m-Pro, m denoting 1, 2 or 3. In these cases the cleavage products obtained start at the N-terminal end with pro and terminate at the C-terminal end with Asp.
Examples of enzymatic cleavages are likewise kno~n, it also being possible to use mod;fied enzymes of improved specificity (cf. C.S. Craik at al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is human pro-insulin it is possible to choose as the sequence Y a pep-tide sequence in which an amino acid which can be cleaved off by trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of the proinsulin, for example Ala-Ser-` 5 1 33632q Met-Thr-Arg, since then the arg;nine-specific cleavage can be carried out ~ith the protease trypsin. If the des;red protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, it is also possible to cleave ~ith factor Xa (Euro-pean Patent Application 0,161,937).
It is also possible in the design of sequence Y-to take ac-count of the synthetic c;rcumstances and to incorporate suitable cleavage sites for restriction enzymes. The DNA
sequence corresponding to the amino acid sequence Y can thus also assume the function of a Linker or adapter.
The fusion protein according to the invention is advanta-geously expressed under the control of the trp operon of E. coli. A DNA segment containing the promoter and operator of the trp operon is no~ commercialLy available. The ex-pression of proteins under the control of the trp operonhas been described many times, for example in European Appli-cation 0,036,776. The induction of the trp operon can be effected by the absence of L-tryptophan and/or the pre-sence of indolyl-3-acrylic acid in the medium.
Under the control of the trp operator there is first trans-cri~?tion of the ~ regian coding for the L-peptide. ~his L-peptide which is composed ~f 14 amino acids, contains L-tryptophan in each of the positians 10 and 11. The rate of protein ~ynthesis of the L-peptide determines ~hether the downstream structural genes are like-~ise translated or ~hether protein synthesis is term;nated.~hen there is a deficiency of L-tryptophan there no~ takes pLace slo~ synthesis of the L-peptide as a result of the ~ow concentration of the tRNA for L-tryptophan, and the follo~ing proteins are synthesized. In contrast, ~hen there are high concentrations of L-tryptophan the corresponding segment of mRNA is rapidly read and termination of protein biosynthesis takes place, since the mRNA assumes a terminator-like structure (C. Yanofsky et al., loc. cit.).
The frequency of translation of a mRNA is great~y influenced by the nature of the nucleotides-in the vicinity of the start codon. Thus, on expression of a fusion protein with the aid of the trp operon, it appears favorable to insert the nucleotides for the first few amino acids of the L-peptide for the start of the structural gene of the fusion protein. In the preferred embodiment of the invention using the trp system the nucleotides of the first three amino acids of the L-peptide (called L'-peptide hereinafter) were chosen as codons for the N-t~rm; n~ 1 amino acids of the fusion protein.
Hence the invention also relates to vectors, preferably plasmids, for the expression of fusion proteins, the DNA of the vectors having the following feature from the 5'-end (in suitable order and in phase): a promoter, an operator, a ribosome binding site and the structural gene for the fusion ~5 protein, the latter containing amino acid sequence I (appendix) upstream of the sequence of the desired protein. Upstream of the structural gene, or as the first triplet of the structural gene, there is located the start codon (ATG) and optionally further codons for other amino acids which are arranged between the start codon and the D'-sequence or between the D~-sequence and the gene for the desired protein. The choice of the DNA
se~uence upstream of the structural gene depends on the amino acid composition of the desired protein, in order to make it possible to cleave the desired protein off from the fusion protein.
It may prove advantageous in the expression of the fusion protein according to the invention to modify individual triplets of the first few amino acids downstream of the ATG
start codon in order to prevent any base-pairing at the level of the mRNA. Modifications of this type are, just as are modifications, deletions or additions of individual amino acids in the D'-protein, familiar to those skilled in the art, and the invention likewise relates to them.
1 33632q - ~ - 7 -Since relatively small plasmids confer several advantages, a preferred embodiment of the invention comprises the e~i-~ mination of a DNA segment with the structural gene for tetra-cycline resistance from pLasmids derived from pBR 322. It is advantageous to delete the segment from the HindIII
restriction site at position 29 to the PvuII restriction site at position 2066. It is particularly advantageous to delete a DNA segment which is even somewhat larger from the plasmids according to the invention, by making use of the PvuII restriction site at the start (in the direction of reading) of the trp operon (which is located in a non-essential part). It is thus possible to carry out direct ligation of the resulting large fragment with the two PvuII
restriction sites. The resulting plasmid, which has been shortened by about 2 kbp, effects an increase in expres-sion, and this is possibly attributable to an increased copy number in the host cell.
The invention is illustrated in detail in the examples which follow.
Example 1 a) Chromosomal E. coli DNA is cut with Hinf I, and the 492 bp fragment which contains the promoter, operator, the struc-tural gene of the L-peptide, the attenuator and the codons for the first six amino acids of the trp-E structural gene from the trp operon is isolated. This fragment is filled in with deoxynucleotide triphosphates with the aid of Klenow polymerase, linked at both ends to an oligonucleo-tide which contains a recognition site for HindIII and is then cut with HindlII~ The HindIII fragment thus obtained is Ligated into the HindIII restriction site of pBR 322.
This results in the plasmid ptrpE2-1 (J.C. Edmann et al., Nature 291 (1981) 503-506). This is converted into the plasmid ptrpL1 as described.
- o t 336329 By use of the synthetic oligonucleotides (N1) and (N2) 5' CGA ~AA TGA AAG CAA AGG 3' (N1) 5' CCT TTG CTT TCA TTG T 3' (N2) which hybridize to th~ double-strand~d ~ligo--nucleotide tN3) 5' CGA CAA TGA AAG CAA AGG 3' 3' T GTT ACT TTC GTT TCC 5' the DNA sequence for the first three amino acids of the L-peptide is incorporated, and a restriction site (StuI) for the insertion of further DNA is formed, in the ClaI
site of the plasmid ptrpL1 (Figure 1). The plasmid ptrpL1 is reacted with the enzyme CLaI in accordance with the manufacturer's instructions, and the mixture is extracted with phenol and the DNA is precipitated with ethanol. The opened plasmid is reacted with al-kaline phosphatase from E. coli to remove the phosphate groups at the 5'-ends. The synthetic nucleotides are phosphorylated at the 5'-ends and are inserted, using T4 DNA ligase, into the opened pLasmid which has been treated with phosphatase. After the li~ase reaction is complete, transformation into E. coli 294 and seLec-tion of the transformants by Amp resistance and the pre-sence of a StuI restriction site are carried out.
About 80Z of the resulting clones had the expected re-striction site ; the nucLeotide sequence depicted in Figure 1 was confirmed by sequence analysis. The plasmid pH120/14 which contains downstream of the ribosome bind-ing site for the L-peptide the nucleotide triplets for the first three amino acids of the L-peptide (L'-peptide), followed by a StuI site which in turn permits the in-sertion of further DNA and thus aLlows the formation of fusion proteins having the first three amino acids of - 9 _ 1 336 32q the L-peptide, is obtained.
b) The example of the oligonucleot;de (N1) employed above ;s used below to ;llustrate the chem;cal synthes;s of such oligonucleot;des:
The method of M.J. Ga;t et al., NucLe;c Acids Research 8 (1980) 1081-1096 is used to bond covalently the nu-cleos;de at the 3'-end, that ;s to say guanos;ne in the present case, to a glass bead support (CPG ~=controlled pore glass) LCAA (=long-chain alkylam;ne) supplied by Pierce) via the 3'-hydroxyl group. This entails the guanosine being reacted as the N-2-;sobutyryl 3'-0-succ;nyl 5'-d;methoxytrityl ether ~;th the modified sup-port in the presence of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyr;dine, there being acylation of the amino radical of the long-chain amine on the support by the free carboxy~ group of the succinyl radical.
In the subsequent steps ;n the synthesis the base com-ponent is used as the dialkylamide or chlor;de of the mono-methyl ester of the 5'-0-d;methoxytr;tylnucLeoside-3'-phosphorous acid, the adenine being in the form of the N6-benzoyl compound, the cytosine being in the form of the N4-benzoyl compound, the guanine being in the form of the N2-isobutyryL compound, and the thymine, ~hich contains no am;no group, being without a protective group.
40 mg of the support containing 1 ~mol of bound guano-s;ne are treated success;vely ~;th the follow;ng agents:
a) Methylene chlor;de, b) 10% tr;chloroacetic acid ;n methylene chloride, c) methanol, d) tetrahydrofuran, e) acetonitriLe, f) 15 ~mol of the appropriate nucleoside phosphite and -70 ~mol of tetrazole in 0.3 ml of anhydrous aceto-nitriLe ~5 minutes), 9) 20% acetic anhydride in tetrahydrofuran containing 4QX lutidine and 10~ dimethylaminopyridine (2 minutes), S h) tetrahydrofuran, i) tetrahydrofuran containing 20X water and 40X lutidine, j) 3% iodine in collidine/water/tetrahydrofuran in the ratio by vo~ume 5:4:1, k) tetrahydrofuran and L) methanol.
The term "phosphite" in this context is defined as the monomethyl ester of the deoxyribose-3'-monophosphorous acid, the third valency being saturated by chlorine or a tertiary amino group, for example a diisopropylamino radical. The yields from the indiv;dual steps in the synthesis can be determined after each detritylation reaction b) by spectrophotometry by measurement of the absorption of the dimethoxytrityl cation at a wavelength of 496 nm.
Once the synthesis of the oligonucleotide is complete, the methyl phosphate protective groups on the oligomer are cleaved off by use of p-thiocresol and triethylamine.
The oligonucleotide is then detached from the solid sup-port by treatment with ammonia for 3 hours. Treatment of the oligomers with concentrated ammonia for 2 to 3 days quantitatively cleaves off the amino protective groups on the bases. The crude product thus obtained is purified by high pressure liquid chromatography (HPLC) or by polyacrylamide gel electrophoresis.
The other oligonucLeotides are also synthesized entirely correspondingly.
c) The plasmid ptrpES-1 (R. A. Hallewell et al., Gene 9 (1980) 27-47) is reacted with the restriction enzymes HindIII and Sa~I in accordance with the manufacturer's ~ 1 33632~
instructions, and the DNA fragment of about ~20 bp is removed. The synthetic oligonucleotides ~N4) and (N5) 5' AGC TTC CAT GAC GCG T 3' (N4) 5' ACG CGT CAT GGA 3' (N5) .
S are phosphorylated, incubated together at 37C and, by use of DNA ligase, added onto the blunt-ended DNA
for proinsuLin t~. ~etekam et al., Gene 19 (1982) 179-183). After reaction with HindIII and SalI, the pro-insulin DNA ~hich has now been extended is covalently incorporated into the opened plasmid using the enzyme T4 DNA ligase (Figure 2), this producing the plasmid pH106/4.
The plasmid pH106/4 is first reacted once more ~ith SalI, the overlapping ends are filled in with KLenow polymerase to give blunt ends and the product is then incubated with the enzyme MstI. A DNA fragment of about S00 bp which contains the entire part coding for proinsuLin and a segment of about 210 bp of the D-protein from the trp operon of E. coLi is isoLated.
The DNA fragment is bLunt-ended and is inserted into the StuI site of the plasmid pH120/14, thus producing the plasmid pH154/25 (Figure 3). This is suitabLe for the expression of a fusion protein under the contro~
of the trp operon, in which the amino ac;d sequence Ala-Ser-Met-Thr-Arg is Located after the L'- and D'-peptide and is foLLowed by the amino acid sequence of proinsuLin.
Example 2 The plasmid pH154/25 (Figure 3) is reacted with the re-striction en2ymes BamHI and XmaIII. The protruding ends are filled in w;th KLenow poLymerase and linked using T4 DNA ligase. This results in the plasm;d pH254 (F;gure 4) 1 3~6329 which is suitable for the expression of a fusion protein having the amino acid sequence L', D'-proinsulin under the control of the trp promoter~ The plasmid is somewhat smal-ler than pH154/25, which may be an advantage.
Example 3 lncubation of the plasmid pH254 (Example 2; Figure 4) with the restriction enzymes MluI and SalI is carried out to liberate a DNA segment of 280 bp, and this is removed. The remainder of the plasmid is converted with Klenow poly-merase into the blunt-ended form and is covalently cyclized again with DNA ligase. This results in the plasmid pH255 (Figure 4) which is suitable for the insertion of a struc-tural gene into one of the restriction sites MluI, SalI
and EcoRI. The formation of a fusion protein with the L', D'-protein is carried out under inducing conditions. Of course, it is possible to insert further restriction sites into the plasmid pH255 by suitable linkers.
Example 4 The plasmid pH154/25 (Figure 3) is incubated with the en-zymes MluI and EcoRI, and the liberated DNA fragment (about 300 bp) is removed. The remainder of the plasmid is filled in with Klenow polymerase. Ring closure is effected by the action of DNA ligase. The resulting pLasmid pH256 (Figure 5) can be used for insertion of structural genes into the EcoRI site.
Example 5 Deletion of a 600 bp fragment from the plasmid pH256 (Example 4; Figure 5) using the restriction enzymes BamHI
and NruI results in the plasmid pH257 (Figure 5). For this purpose, pH256 is first incubated with BamHI and blunt ends are generated with Klenow polymerase. After incubation with NruI and removal of the 600 bp fragment the formation ~ 1 336329 of pH257 is effected after incubation ~ith DNA ligase.
Example 6 Insertion of the lac repressor (P.J. Farabaugh, Nature 274, (1978) 765-769) into the plasmid pKK 177-3 (A~ann et al., S Gene 25 (1983) 167) resu~ts in the p~asmid pJF1~8. This is reacted with EcoRI and SalI, and the remainder of the plasmid is isolated.
A fragment about 495 bp in size is obtained from the plas-mid pH106/4 (Figure 2) by the action of SalI and incubation ~ith MstI.
The oligonucleotides $N6) and (N7) obtained by synthesis 5' ACG AAT TCA TGA AAG CAA AGG 3' (N6) 5' CCT TTG CTT TCA TGA ATT CGT 3' (N7) are phosphorylated and, using DNA ligase, added onto the blunt-ended DNA fragment. Reaction ~ith EcoRI and Sa~I
liberates oYerlappin~ ends which permit ~igation into the opened plasmid pJF118.
After transformation into E. coli 294 of the hybrid plasmid which has thus been obtsined, the correct clones are selec-ted on the basis of the size of the restriction fragments.This plasmid is called pJ120 (Figure 6).
The expression of the fusion protein is carried out in shaken flasks as follo~s:
An overnight culture in LB medium (J.H. Miller, Experi~ents 2~ in Molecular Genetics, Cold Spring Harbor Laboratory, 1972), containing 50 ~g/ml ampicillin, of E. coli 294-transformants which contain the plasmid pJ120 is used to set up a fresh culture in the ratio of about 1:100, and the growth is follo~ed by measurement of the OD. ~hen the OD is O.S, isopropyl B-D-galactopyranoside (IPTG) is added to the culture in an amount such that its concentration is 1 mM, and the bacteria are removed by centrifugation after 150 to 180 minutes. The bacteria are boiled for 5 minutes in a buffer mixture (7M urea, 0.1X SDS, 0.1 M sodium phosphate, pH 7.0), and samples are applied to a SDS gel e~ectrophoresis plate. After the electrophoresis, the bacteria which con-tain the plasmid pJ120 provide a protein band vhich corres-ponds to the size of the expected fusion protein and whichreacts ~ith antibodies against insulin. After the fusion protein has been isolated the expected proinsulin derivative can be liberated by cleavage with cyanogen bromide. After disruption of the bacteria (French Press;
(R)Dyno-mill ) and centrifugation, the L', D'-proinsu-lin fusion protein is located in the sediment, so that con-siderable amounts of the other proteins can nou be removed ~ith the supernatant.
The stated induction conditions apply to shake cultures;
for Larger fermentations it is advantageous to modify the OD
values accordingly and, ~here appropriate, to vary the IPTG
concentrations slightly.
Example 7 An overnight culture in LB medium containing SO ~g/ml ampi-cillin is prepared from E. co~i 294 transformants which contain the plasmid pH154/2S (Figure 3) and the next morn-ing ;s diluted in the ratio of about 1:100 in M9 medium (J. H. Miller, Loc. cit.) containing 2000 ~g/ml casamino acids and 1 ~g/ml thiamine. ~hen the OD = O.S indolyl-3-acryl;c acid is added so that the final concentration is15 ~g/ml. After incubation for 2 to 3 hours, the bacteria are removed by centr;fugation. SDS gel electrophoresis shows a very pronounced protein band ~hich is at the place ex-pected for the fus;on protein and which reacts ~ith anti-bodies against insulin. After disruption of the bacteriaand centrifugation, the L', D'-proinsu~in fusion protein - ~ - 15 -is located in the sediment so that, again, considerable amounts of the other proteins are no~ removed with the supernatant.
In the present case too, the stated induction conditions apply to shake cultures. Fermentations in larger volumes require altered concentrations of casamino acids or addi-tion of L-tryptophan.
Example 8 The plasmid pH154/25 (Figure 3) is opened ~ith EcoRI, and the protruding DNA single strands are filled in ~ith Klenow polymerase. The DNA thus obtained is incubated ~ith the enzyme MluI, and the DNA coding for insulin is cut out of the plasmid. Separation by gel electrophoresis is carried out to remove this fragment from the remainder of the plas-mid, and the remainder of the plasmid is isoLated.
The plasmid sho~n in Figure 3 of German Offenlegungsschrift3,429,430 (European Patent Application A1 0,171,024) is reacted with the restriction enzymes AccI and SaLI, and the DNA fragment containing the hirudin sequence is removed.
After the protruding ends of the SalI restriction site have been filled in with Klenow polymerase, the DNA segment is ligated ~ith the synthetic DNA of the formula (N8) Met Thr 5' CCC ACG CGT ATG ACG T 3' 3' GGG TGC GCA TAC TGC ATA 5' (N8) The ligation product is incubated with MluI. After the en-zyme has been inactivated at 65C, the DNA mixture is treated ~ith bovine alkaline phosphatase at 37C for one hour.
This is followed by removal of the phosphatase and the re-striction enzyme from the mixture by extraction with phenol,and the DNA is purified by ethanol precipitation. The DNA
~hich has thus been treated is inserted using T4 ligase -~ 1 336329 ;nto the opened remainder of the plasmid pH154/25, this resulting in the plasmid pK150 which has been characterized by restriction anaLysis and DNA sequencing by the method of Maxam and Gilbert (Figure 7).
Example 9 E. coli 29~ bacter;a which contain the plasmid pK150 (Fi-gure 7) are cultured in LB medium containing 30 to 50 ~g/ml ampicillin at 37C overnight. The culture is diLuted in the ratio of 1:100 ~ith M9 medium which contains 2000 ~g/ml casamino acids and 1 ~g/ml thiamine, and the mixture is incubated at 37C, mixing continuously. ~hen the OD600= 0.5 or 1, indolyl-3-acrylic acid is added to a final concentration of 15 ~/ml, and the mixture is incubated for 2 to 3 hours or 16 hours respect;vely. The bacteria are then removed by centrifugation and disrupted in 0.1 M
sodium phosphate buffer tpH 6.5) under pressure. The spar-ingly soluble proteins are removed by centrifugation and analyzed by SDS polyacryLamide gel electrophoresis. It emerges that cells whose trp operon has been induced con-tain in the region below 20,000 Daltons but above14,000 Da~-tons a new protein ~hich is not found in non-induced cells.
After the fusion protein has been isolated and reacted with cyanogen bromide hirudin is liberated.
Example 10 The constructs described hereinafter permit the introduc-tion, upstream of the 5'-end of the trp-D sequence, of DNA
sequences which contain as man~ recognition sites for various restriction enzymes as possible in order to incorporate the trp-D sequence into as many as possible of the wide variety of prokaryoctic expression systems.
The plasmids pUC12 and pUC13 (Pharmacia P-L Biochemicals, 5401 St. Goar: The Molecular Biology Catalogue 1983, Ap-~ - 17 - 1 336329 pendix, p. 89) contain ~ polylinker se~uence, it being the intention to ;nsert in the pl~sm;d pUC13, bet~een the restric-tion cleavage sites for XmaI ~nd SacI, the Mstl-HindIII trp fr~gment from the pl~smid p106/4 (Figure 2) ~hich is fused vith the HindIII-hirudin-SacI fragment from the plasmid pK150 ~Figure 7).
For this purpose, the DNA of the pl~snid pUC13 is first treated ~ith the restriction enzyme XmaI. The ends of the ~inearized plasmid are fil~ed in by ~eans of the Kleno~
polymerase reaction. After ethanol precipitation, the DNA
is treated ~ith the enzy~e SacI and is ~g~in precipitated fro0 the reaction mixture ~ith ethanol. The DNA is no~
reacted in ~n aqueous ligation ~ixture Yith the MstI-HindIlI trp-D fragment isol~ted from plasmid pH106/4 and ~ith the HindIII-Sac~ hirudin fra~ment isolated from the plas~id pK150, ~nd T4 DNA ligase.
The plasmid pK160 thus obtained no~ cont~ins, i~mediately upstream of the trp-D sequence, aultiple restriction en-zyme recognition sequence ~hich e~br~ces restriction sites for the enz~nes XmaI, S~aI, BamHI, XbaI, HincII, SalI, AccI, PstI ~nd HindIII. Furthermore, ~n EcoRI restriction site is generated do~nstre-m of the 3'-end of the hirudin se-~uence in this construction (Figure 8).
Example 11 The plasmid pH131/5 is prepared as follows:
The plas~id ptrpL1 is opened with ClaI and ligated with the synthe-tically prepared, self-comPlement~ry oligonucleotide (N9) 5' pCGACCATGGT 3'; ~N9) .
~he plasmid pH131/5 (Figure 9) thus obtained is opened at the restriction site, vhich has been introduced in this 0anner, - ~ ~ 336329 _ - 18 -~ for the restriction enzyme NcoI, and the resulting pro-truding single-stranded ends are filled in by means of the - KLenov polymerase reaction. The linearized, blunt-ended DNA is now cut w;th the enzyme EcoRI, and the lar~er of the two resuLting DNA sequences is separated from the smal-ler sequence by ethanol precipitation. The rema;nder of the pLasm;d DNA of the plasmid pH131/5 thus obtained is now Ligated with a fragment from pK160 coding for trp-D'-hirudin by reaction vith T4 ligase. This fragment is cleaved out of the p~asm;d pK160 by opening the plasm;d w;th HincII and EcoRI. The fragment is removed from the remainder of the plasmid by gel eLectrophoresis, and ;s then eluted from the gel material. The Ligation product ;s transformed ;nto E. coLi K12. The clones containing plasmid DNA are ;solated and characterized by restriction anaLysis and DNA sequence analysis. The plasmid pK170 thus obtained contains fused onto the trp operator a DNA
sequence which codes for Met-Asp-Ser-Arg-GLy-Ser-Pro-Gly-trp-D'-(hirudin~ (Figure 9).
ExampLe 12 The pLasm;d pJF118 (Example 6) is opened with EcoRI, and the protruding DNA ends are converted into bLunt ends by means of the KLenow polymerase reaction. The DNA thus treated is then cut with the enzyme SalI, and the short EcoRI-SaLI fragment is removed by gel eLectrophoresis.
The pLasmid pK 170 (Exsmple 11) is cleaved with NcoI, and the protruding ends are converted into bLunt ends using Klenov polymerase. The plasmid DNA is removed from the reaction mixture by ethanol precipitat;on and ;s treated with the enzymes HindIII and BamHI. Two of the resuLting fragments are ;solated, namely the NcoI (w;th fiLled-in end)-trp-D'-HindIII fragment and the H;ndIII-h;rud;n-~amHI
fragment (German Offenlegungsschr;ft 3,429,430). The two fragments are isolated after separation by geL eLectro-phoresis.
-- 1 33632~
In addition, the BamHI-SalI-HirudinII fragment shown in Figure 2 of German Offenlegungsschrift 3,429,430 is iso-lated. In a ligation reaction the four fragments, namely the remainder of the plasmid pJF 118, the NcoI-trp-D'-HindIII fragment, the HindIII-hirudin-BamHI fragment and the hirudinII fragment, are now reacted together and the resuLting plasmid pK 180 (Figure 10) is transformed into E. col; K12-W 3110. Correct plasmids are shown by it be;ng possible to detect an EcoRI-trp-D'-hirudin-SalI fragment in the pLasmid DNA. The trp-D'-hirudin sequence is now attached to the tac promoter. The fusion protein is ex-pressed as in Example 6.
Example 13 The plasmids derived from pBR 322, such as pH120/14 (Ex-ample 1, Figure 1), pH154/25 (ExampLe 1, Figure 3), pH256 (Example 4, Figure 5), pK150 (Example 8, Figure 7) and pK170 (Example 11, Figure 9), have - on the figures in the clockwise direction - between the start codon of the fusion protein and the next HindIII site (corresponding to HindIII
at position 29 in pBR322) an additional PvuII site ;n the region of the fragment which contains the trp promoter and operator, but outside the promoter region.
It has now been found that by deletion of the DNA segment which is bounded by the PvuII site described and the PvuII
site which corresponds to position 2066 in pBR322, the yield of a cloned protein (or fusion protein) is dis~inctly in-creased~
The shortening of the p~asmid pH154/25 to give pH154/25*
is described by way of example hereinafter, it being pos-s;ble for this to be effected correspondingly for the other plasmids mentioned above (the shortened plasmids likewise being identified by 3n asterisk):
pH154/25 is reacted with PvulI ~in accordance with the manu--1 33632~
facturer's instructions), resulting ;n three fragments:
t Fragment 1: From the PvuII restriction site of the proin-sulin gene to the PvuII restriction site cor-respond;ng to position 2066 in pBR322, Fragment 2: from the PvuII restriction site near the trp promoter to the PvuII site of the proinsulin gene and Fragment 3: from the PvuII site near the trp promoter fragment to the PvuII site corresponding to position 2066 in pBR322.
The fragments can be separated by electrophoresis on agarose and then isolated (Maniatis eb al., Molecular Cloning, Cold Spring Harbor, 1982).
The fragments 1 and 2 are joined under blunt end conditions using the enzyme T4 DNA ligase. Transformation into E. coli 294 is followed by testing for those colonies ~hich contain a plasmid ~ith the complete proinsulin sequence and thus have the fragments in the desired order. The pLasmid pH154/25* is depicted in Figure 11.
A distinct increase in the proportion of fusion protein is observed on expression, ~hich is carried out as described in the preceding examples.
Example 14 The plasmid pH 154/25* ~Example 13, Figure 11) is digested ~ith HindIII and SalI, and the small fragment (having the proinsulin sequence) is separated off by gel eLectrophore-sis. The large fragment is isolated and ligated ~ith the synthetic DNA (N10) (Ala) 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
The plasmid pInt13 (Figure 12) is produced.
The DNA (N10) codes for an amino acid sequence which con-tains several cleavage sites for chemical cleavage:
a) Met for cyanogen bro-ide, b) Trp for N-bromosuccinimide (NBS or BSI) c) Asp-Pro for proteolytic cleavage, the upstream Glu ad-ditionally weakening the Asp-Pro bond to~ards the action of acids.
The introduction of this HindlII-Sa~l-linker (N10) into the reading frame of a coded polypeptide thus allo~s the options which have been mentioned for chemical cleavage off, de-pending on the amino acid sequence of the desired prote;n and on its sensitivity to the cleaving agents.
The figures are not to scale~
' ~ - 22 - 1 336329 Amino ac;d sequence I
23) Ser Asn Gly His Asn Val Val Ile Tyr Arg Asn His Ile Pro Ala Gln Thr Leu Ile Glu Arg Leù A~a Thr Met Ser Asn Pro Val Leu Met Leu Ser Pro Gly Pro Gly VaL Pro Ser Glu Ala Gly Cys Met Pro Glu Leu Leu Thr Arg Leu Arg Gly Lys Leu Pro ILe Ile Gly ILe Cys Leu Gly (93) His Gln ALa Ile Val Glu ALa
Fusion proteins, a process for their preparation and their use In the preparation by genetic engineering methods of rela-tively small eukaryotic proteins with a molecul~r ~eight up to about 15000 Daltons the yield obtained in bacteria is frequently only small. It is presumed that the proteins which are formed are rapidly degraded by proteases intrin-sic to the host. For this reason, proteins of this type - are advantageously prepared as fusion proteins, in particu-Lar having a portion of protein intrinsic to the host, which is then cleaved off.
It has now been found that a segment composed of only about 70 amino acids of the D-protein from the trp operon of E.
coli is particularly suitable for the formation of fusion proteins, specifically in the region of the sequence of amino acids 23 to 93 (C. Yanofsky et al., NucLeic Acids Res.
9 (1981)6647), also caLled "D'-peptide" hereinafter. Be-tween the carboxyl terminal end of this peptide and of the amino acid sequence of the desired eukaryotic protein there is a sequence comprising one or more genetically codabLe Dmino acids which permits the desired protein to be cleaved off chemicalLy or enzymatically. In preferred em-bodiments of this invention, the amino terminal end ;s foL-lowed by a short amino acid sequence composed of Lys-Ala, optionally followed by a sequence of 1 to 10, in particular 1 to 3, other genetically codable amino acids, preferably by two amino acids, in particular by Lys-Gly.
Hence the invention relates to a fusion protein of the general formula Met-Xn-D'-Y-Z
in which n is zero or 1, i, ~ - 2 - l 33~329 X is a sequence of 1 to 12 gen~t~cally codable amino aC~s, preferably Lys-Ala, D' is a sequence of about 70 amino acids in the region o~f 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 representing the desired final protein.
The present invention will now be described in association with preferred embodiments with reference to accompanying drawings, in which:
Figure 1 shows a process for preparing plasmid pH120/14, which is suitable ~or the expression of a fusion protein having the first three amino acids of the L-peptide.
Figure 2 shows a process for preparing plasmid pH106/4 coding for proinsulin.
Figure 3 shows a process for preparing plasmid pH154/25, which is suitable for the expression of a fusion protein under the control of the trp operon, in which the amino acid sequence Ala-Ser-Met-Thr-Arg is located after the L'- and D'-peptide and is followed by the amino acid sequence of proinsulin.
Figure 4 shows a process for preparing plasmid pH254, which is suitable for the expression of a fusion protein having the amino acid sequence L', D~-proinsulin under the control of the trp promoter, and a process for preparing plasmid pH255 which is suitable for the insertion of a structural gene into one of the restriction sites MluI, SalI and EcoRI.
Figure 5 shows a process for preparing plasmid pH256 which is suitable for the insertion of structural genes into the EcoRI
site, and a process for preparing plasmid pH257.
1~
~ - 2a .,.
Figure 6 shows a process for ~ ~ ing plasmid pJ1~ -coding for proinsulin.
Figure 7 shows a process for preparing plasmid pK150 coding for hirudin.
Figure 8 shows a process for preparing plasmid pK160, which contains, immediately upstream of the trp-D sequence, a multiple restriction enzyme recognition sequence which embraces restriction sites for the enzymes XmaI, SmaI, BamHI, XbaI, HincII, SalI, AccI, PstI and HindIII, and an EcoRI restriction site downstream of the 3'-end of the hirudin sequence.
Figure 9 shows a process for preparing plasmid pK170 which contains a DNA sequence which codes for Met-Asp-Ser-Arg-Gly-Ser-Pro-Gly-trp-D'-(hirudin) fused onto the trp operator.
Figure 10 shows a process for preparing pK180 coding for hirudin.
Figure 11 shows a process for preparing pH154/25 * coding for proinsulin.
Figure 12 shows a process for preparing plasmid pint 13 coding for an amino ac`id sequence allowing chemical cleavage by cyanogen bromide, n-bromosuccinimide or acids.
~ ~ - 2b ' .. , , ;
Further aspects of the invention and preferred embodiments are described here;nafter and defined in the patent claims.
Of course, it i~ advantageous if the undesired portion (in-trinsic to the host) of the fusion protein is as small as possible since then the cell produces only little "ballast"
and hence the yield of desired protein is high. Further-more, when the undesired portion is cleaved off, fewer by-products are produced, which fac;litates working up~ A
factor opposing this is that the (assumed) "protective function" of the undesired portion is to be expected only above a certain size. It has now emerged, surprisingly, that the segment chosen according to the ;nvention from the D-protein fulfils this task although it contains only about 70 amino acids.
In many cases, especially in the preferred embodiment in which X represents Lys-Ala or contains this sequence at the ~-terminal end, the fusion protein formed is insoluble.
The latter can easiLy be separated from the solubLe pro-teins, which great~y facilitates the working up and in-creases the y;eld. The formation of an insolubLe fusion prote;n is surpr;s;ng since, on the one hand, the bacterial portion of only about 70 amino acids ;s quite small and, on the other hand, it is a constituent of a protein which is present in solution in the host cell~
"About 70 amino ac;ds in the region of the sequence of amino _ 3 _ 1336329 acids 23 to 93 of the D-peptide" means that it is possible to carry out, in a manner known per se, variations, that is to say it is poss;ble for individual amino acids to be deleted, replaced or exchanged without this significantly changing the properties of the fusion proteins according to the invention. The invention likewise relates to vari-ations of this type.
The desired eukaryotic protein is preferably a biologicalLy active protein, such as a hirudin, or a precursor of a pro-tein of this type, such as human proinsulin.
The fusion protein is obtained by expression in a suitablesystem, and in the particularly preferred embodiment is, after disruption of the host cells, isolated from the sedi-ment in which it is concentrated owing to its sparing solu-bility. Hence it is easy to separate it from the solubLeconstituents of the cell.
Suitable host cells are all those for which expression sys-tems are known , that is to say mammalian cells and micro-organisms, preferably bacteria, in particular E. coli since, after all, the bacterial portion of the fusion protein is a protein intrinsic to the host E. coli.
The DNA sequence which codes for the fusion protein accord-ing to the invention is incorporated, in a known manner, into a vector which ensures satisfactory expression in the selected expression system.
In bacterial hosts, it is expedient to choose the promoter and operator from the group comprising Lac, Tac, PL or PR of the phage ~, hsp, omp or a synthetic promoter, such as are described, for example, in German Offenlegungs-schrift 3,430,683 (European Patent Application 0,173,149)~
A particularly suitable vector is one which contains the following elements of the trp operon of E. coli: the pro-4moter, the operator and the ribosome binding site of the L-peptide. It is particularly advantageous for the first three amino acids of this L-peptide to follow in the cod-ing region, and then to be followed by a short amino acid sequence and the amino acids 23 to 93 of the D-protein in the trp operon.
The intermediate sequence Y which makes it possible to cleave off the desired polypeptide depends on the compo-sition of this desired peptide: for example, if this con-tains no methionine it is possible for Y to denote Metand then chemical cleavage with cyanogen bromide is car-ried out. If there is cysteine at the carboxyl terminal end in the connecting member Y, or if Y represents Cys, then cysteine-specific enzymatic cleavage or chemical cleav-age, for example after specific S-cyanylation, can be car-ried out. If there is tryptophan at the carboxyl terminal end of the bridging member r, or if Y represents Trp, then chemical cleavage with N-bromosuccinimide can be carried out. If Y represents Asp-Pro, then proteolytic cleavage can be carried out in a manner known per se (D. Piszkie~icz et al., Biochemical and Biophysical Research Communications 40 (1970) 1173-1178). The Asp-Pro bond can, as has been found, be made even more acid-labile if Y is (Asp)m-Pro or Glu-(Asp)m-Pro, m denoting 1, 2 or 3. In these cases the cleavage products obtained start at the N-terminal end with pro and terminate at the C-terminal end with Asp.
Examples of enzymatic cleavages are likewise kno~n, it also being possible to use mod;fied enzymes of improved specificity (cf. C.S. Craik at al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is human pro-insulin it is possible to choose as the sequence Y a pep-tide sequence in which an amino acid which can be cleaved off by trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of the proinsulin, for example Ala-Ser-` 5 1 33632q Met-Thr-Arg, since then the arg;nine-specific cleavage can be carried out ~ith the protease trypsin. If the des;red protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, it is also possible to cleave ~ith factor Xa (Euro-pean Patent Application 0,161,937).
It is also possible in the design of sequence Y-to take ac-count of the synthetic c;rcumstances and to incorporate suitable cleavage sites for restriction enzymes. The DNA
sequence corresponding to the amino acid sequence Y can thus also assume the function of a Linker or adapter.
The fusion protein according to the invention is advanta-geously expressed under the control of the trp operon of E. coli. A DNA segment containing the promoter and operator of the trp operon is no~ commercialLy available. The ex-pression of proteins under the control of the trp operonhas been described many times, for example in European Appli-cation 0,036,776. The induction of the trp operon can be effected by the absence of L-tryptophan and/or the pre-sence of indolyl-3-acrylic acid in the medium.
Under the control of the trp operator there is first trans-cri~?tion of the ~ regian coding for the L-peptide. ~his L-peptide which is composed ~f 14 amino acids, contains L-tryptophan in each of the positians 10 and 11. The rate of protein ~ynthesis of the L-peptide determines ~hether the downstream structural genes are like-~ise translated or ~hether protein synthesis is term;nated.~hen there is a deficiency of L-tryptophan there no~ takes pLace slo~ synthesis of the L-peptide as a result of the ~ow concentration of the tRNA for L-tryptophan, and the follo~ing proteins are synthesized. In contrast, ~hen there are high concentrations of L-tryptophan the corresponding segment of mRNA is rapidly read and termination of protein biosynthesis takes place, since the mRNA assumes a terminator-like structure (C. Yanofsky et al., loc. cit.).
The frequency of translation of a mRNA is great~y influenced by the nature of the nucleotides-in the vicinity of the start codon. Thus, on expression of a fusion protein with the aid of the trp operon, it appears favorable to insert the nucleotides for the first few amino acids of the L-peptide for the start of the structural gene of the fusion protein. In the preferred embodiment of the invention using the trp system the nucleotides of the first three amino acids of the L-peptide (called L'-peptide hereinafter) were chosen as codons for the N-t~rm; n~ 1 amino acids of the fusion protein.
Hence the invention also relates to vectors, preferably plasmids, for the expression of fusion proteins, the DNA of the vectors having the following feature from the 5'-end (in suitable order and in phase): a promoter, an operator, a ribosome binding site and the structural gene for the fusion ~5 protein, the latter containing amino acid sequence I (appendix) upstream of the sequence of the desired protein. Upstream of the structural gene, or as the first triplet of the structural gene, there is located the start codon (ATG) and optionally further codons for other amino acids which are arranged between the start codon and the D'-sequence or between the D~-sequence and the gene for the desired protein. The choice of the DNA
se~uence upstream of the structural gene depends on the amino acid composition of the desired protein, in order to make it possible to cleave the desired protein off from the fusion protein.
It may prove advantageous in the expression of the fusion protein according to the invention to modify individual triplets of the first few amino acids downstream of the ATG
start codon in order to prevent any base-pairing at the level of the mRNA. Modifications of this type are, just as are modifications, deletions or additions of individual amino acids in the D'-protein, familiar to those skilled in the art, and the invention likewise relates to them.
1 33632q - ~ - 7 -Since relatively small plasmids confer several advantages, a preferred embodiment of the invention comprises the e~i-~ mination of a DNA segment with the structural gene for tetra-cycline resistance from pLasmids derived from pBR 322. It is advantageous to delete the segment from the HindIII
restriction site at position 29 to the PvuII restriction site at position 2066. It is particularly advantageous to delete a DNA segment which is even somewhat larger from the plasmids according to the invention, by making use of the PvuII restriction site at the start (in the direction of reading) of the trp operon (which is located in a non-essential part). It is thus possible to carry out direct ligation of the resulting large fragment with the two PvuII
restriction sites. The resulting plasmid, which has been shortened by about 2 kbp, effects an increase in expres-sion, and this is possibly attributable to an increased copy number in the host cell.
The invention is illustrated in detail in the examples which follow.
Example 1 a) Chromosomal E. coli DNA is cut with Hinf I, and the 492 bp fragment which contains the promoter, operator, the struc-tural gene of the L-peptide, the attenuator and the codons for the first six amino acids of the trp-E structural gene from the trp operon is isolated. This fragment is filled in with deoxynucleotide triphosphates with the aid of Klenow polymerase, linked at both ends to an oligonucleo-tide which contains a recognition site for HindIII and is then cut with HindlII~ The HindIII fragment thus obtained is Ligated into the HindIII restriction site of pBR 322.
This results in the plasmid ptrpE2-1 (J.C. Edmann et al., Nature 291 (1981) 503-506). This is converted into the plasmid ptrpL1 as described.
- o t 336329 By use of the synthetic oligonucleotides (N1) and (N2) 5' CGA ~AA TGA AAG CAA AGG 3' (N1) 5' CCT TTG CTT TCA TTG T 3' (N2) which hybridize to th~ double-strand~d ~ligo--nucleotide tN3) 5' CGA CAA TGA AAG CAA AGG 3' 3' T GTT ACT TTC GTT TCC 5' the DNA sequence for the first three amino acids of the L-peptide is incorporated, and a restriction site (StuI) for the insertion of further DNA is formed, in the ClaI
site of the plasmid ptrpL1 (Figure 1). The plasmid ptrpL1 is reacted with the enzyme CLaI in accordance with the manufacturer's instructions, and the mixture is extracted with phenol and the DNA is precipitated with ethanol. The opened plasmid is reacted with al-kaline phosphatase from E. coli to remove the phosphate groups at the 5'-ends. The synthetic nucleotides are phosphorylated at the 5'-ends and are inserted, using T4 DNA ligase, into the opened pLasmid which has been treated with phosphatase. After the li~ase reaction is complete, transformation into E. coli 294 and seLec-tion of the transformants by Amp resistance and the pre-sence of a StuI restriction site are carried out.
About 80Z of the resulting clones had the expected re-striction site ; the nucLeotide sequence depicted in Figure 1 was confirmed by sequence analysis. The plasmid pH120/14 which contains downstream of the ribosome bind-ing site for the L-peptide the nucleotide triplets for the first three amino acids of the L-peptide (L'-peptide), followed by a StuI site which in turn permits the in-sertion of further DNA and thus aLlows the formation of fusion proteins having the first three amino acids of - 9 _ 1 336 32q the L-peptide, is obtained.
b) The example of the oligonucleot;de (N1) employed above ;s used below to ;llustrate the chem;cal synthes;s of such oligonucleot;des:
The method of M.J. Ga;t et al., NucLe;c Acids Research 8 (1980) 1081-1096 is used to bond covalently the nu-cleos;de at the 3'-end, that ;s to say guanos;ne in the present case, to a glass bead support (CPG ~=controlled pore glass) LCAA (=long-chain alkylam;ne) supplied by Pierce) via the 3'-hydroxyl group. This entails the guanosine being reacted as the N-2-;sobutyryl 3'-0-succ;nyl 5'-d;methoxytrityl ether ~;th the modified sup-port in the presence of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyr;dine, there being acylation of the amino radical of the long-chain amine on the support by the free carboxy~ group of the succinyl radical.
In the subsequent steps ;n the synthesis the base com-ponent is used as the dialkylamide or chlor;de of the mono-methyl ester of the 5'-0-d;methoxytr;tylnucLeoside-3'-phosphorous acid, the adenine being in the form of the N6-benzoyl compound, the cytosine being in the form of the N4-benzoyl compound, the guanine being in the form of the N2-isobutyryL compound, and the thymine, ~hich contains no am;no group, being without a protective group.
40 mg of the support containing 1 ~mol of bound guano-s;ne are treated success;vely ~;th the follow;ng agents:
a) Methylene chlor;de, b) 10% tr;chloroacetic acid ;n methylene chloride, c) methanol, d) tetrahydrofuran, e) acetonitriLe, f) 15 ~mol of the appropriate nucleoside phosphite and -70 ~mol of tetrazole in 0.3 ml of anhydrous aceto-nitriLe ~5 minutes), 9) 20% acetic anhydride in tetrahydrofuran containing 4QX lutidine and 10~ dimethylaminopyridine (2 minutes), S h) tetrahydrofuran, i) tetrahydrofuran containing 20X water and 40X lutidine, j) 3% iodine in collidine/water/tetrahydrofuran in the ratio by vo~ume 5:4:1, k) tetrahydrofuran and L) methanol.
The term "phosphite" in this context is defined as the monomethyl ester of the deoxyribose-3'-monophosphorous acid, the third valency being saturated by chlorine or a tertiary amino group, for example a diisopropylamino radical. The yields from the indiv;dual steps in the synthesis can be determined after each detritylation reaction b) by spectrophotometry by measurement of the absorption of the dimethoxytrityl cation at a wavelength of 496 nm.
Once the synthesis of the oligonucleotide is complete, the methyl phosphate protective groups on the oligomer are cleaved off by use of p-thiocresol and triethylamine.
The oligonucleotide is then detached from the solid sup-port by treatment with ammonia for 3 hours. Treatment of the oligomers with concentrated ammonia for 2 to 3 days quantitatively cleaves off the amino protective groups on the bases. The crude product thus obtained is purified by high pressure liquid chromatography (HPLC) or by polyacrylamide gel electrophoresis.
The other oligonucLeotides are also synthesized entirely correspondingly.
c) The plasmid ptrpES-1 (R. A. Hallewell et al., Gene 9 (1980) 27-47) is reacted with the restriction enzymes HindIII and Sa~I in accordance with the manufacturer's ~ 1 33632~
instructions, and the DNA fragment of about ~20 bp is removed. The synthetic oligonucleotides ~N4) and (N5) 5' AGC TTC CAT GAC GCG T 3' (N4) 5' ACG CGT CAT GGA 3' (N5) .
S are phosphorylated, incubated together at 37C and, by use of DNA ligase, added onto the blunt-ended DNA
for proinsuLin t~. ~etekam et al., Gene 19 (1982) 179-183). After reaction with HindIII and SalI, the pro-insulin DNA ~hich has now been extended is covalently incorporated into the opened plasmid using the enzyme T4 DNA ligase (Figure 2), this producing the plasmid pH106/4.
The plasmid pH106/4 is first reacted once more ~ith SalI, the overlapping ends are filled in with KLenow polymerase to give blunt ends and the product is then incubated with the enzyme MstI. A DNA fragment of about S00 bp which contains the entire part coding for proinsuLin and a segment of about 210 bp of the D-protein from the trp operon of E. coLi is isoLated.
The DNA fragment is bLunt-ended and is inserted into the StuI site of the plasmid pH120/14, thus producing the plasmid pH154/25 (Figure 3). This is suitabLe for the expression of a fusion protein under the contro~
of the trp operon, in which the amino ac;d sequence Ala-Ser-Met-Thr-Arg is Located after the L'- and D'-peptide and is foLLowed by the amino acid sequence of proinsuLin.
Example 2 The plasmid pH154/25 (Figure 3) is reacted with the re-striction en2ymes BamHI and XmaIII. The protruding ends are filled in w;th KLenow poLymerase and linked using T4 DNA ligase. This results in the plasm;d pH254 (F;gure 4) 1 3~6329 which is suitable for the expression of a fusion protein having the amino acid sequence L', D'-proinsulin under the control of the trp promoter~ The plasmid is somewhat smal-ler than pH154/25, which may be an advantage.
Example 3 lncubation of the plasmid pH254 (Example 2; Figure 4) with the restriction enzymes MluI and SalI is carried out to liberate a DNA segment of 280 bp, and this is removed. The remainder of the plasmid is converted with Klenow poly-merase into the blunt-ended form and is covalently cyclized again with DNA ligase. This results in the plasmid pH255 (Figure 4) which is suitable for the insertion of a struc-tural gene into one of the restriction sites MluI, SalI
and EcoRI. The formation of a fusion protein with the L', D'-protein is carried out under inducing conditions. Of course, it is possible to insert further restriction sites into the plasmid pH255 by suitable linkers.
Example 4 The plasmid pH154/25 (Figure 3) is incubated with the en-zymes MluI and EcoRI, and the liberated DNA fragment (about 300 bp) is removed. The remainder of the plasmid is filled in with Klenow polymerase. Ring closure is effected by the action of DNA ligase. The resulting pLasmid pH256 (Figure 5) can be used for insertion of structural genes into the EcoRI site.
Example 5 Deletion of a 600 bp fragment from the plasmid pH256 (Example 4; Figure 5) using the restriction enzymes BamHI
and NruI results in the plasmid pH257 (Figure 5). For this purpose, pH256 is first incubated with BamHI and blunt ends are generated with Klenow polymerase. After incubation with NruI and removal of the 600 bp fragment the formation ~ 1 336329 of pH257 is effected after incubation ~ith DNA ligase.
Example 6 Insertion of the lac repressor (P.J. Farabaugh, Nature 274, (1978) 765-769) into the plasmid pKK 177-3 (A~ann et al., S Gene 25 (1983) 167) resu~ts in the p~asmid pJF1~8. This is reacted with EcoRI and SalI, and the remainder of the plasmid is isolated.
A fragment about 495 bp in size is obtained from the plas-mid pH106/4 (Figure 2) by the action of SalI and incubation ~ith MstI.
The oligonucleotides $N6) and (N7) obtained by synthesis 5' ACG AAT TCA TGA AAG CAA AGG 3' (N6) 5' CCT TTG CTT TCA TGA ATT CGT 3' (N7) are phosphorylated and, using DNA ligase, added onto the blunt-ended DNA fragment. Reaction ~ith EcoRI and Sa~I
liberates oYerlappin~ ends which permit ~igation into the opened plasmid pJF118.
After transformation into E. coli 294 of the hybrid plasmid which has thus been obtsined, the correct clones are selec-ted on the basis of the size of the restriction fragments.This plasmid is called pJ120 (Figure 6).
The expression of the fusion protein is carried out in shaken flasks as follo~s:
An overnight culture in LB medium (J.H. Miller, Experi~ents 2~ in Molecular Genetics, Cold Spring Harbor Laboratory, 1972), containing 50 ~g/ml ampicillin, of E. coli 294-transformants which contain the plasmid pJ120 is used to set up a fresh culture in the ratio of about 1:100, and the growth is follo~ed by measurement of the OD. ~hen the OD is O.S, isopropyl B-D-galactopyranoside (IPTG) is added to the culture in an amount such that its concentration is 1 mM, and the bacteria are removed by centrifugation after 150 to 180 minutes. The bacteria are boiled for 5 minutes in a buffer mixture (7M urea, 0.1X SDS, 0.1 M sodium phosphate, pH 7.0), and samples are applied to a SDS gel e~ectrophoresis plate. After the electrophoresis, the bacteria which con-tain the plasmid pJ120 provide a protein band vhich corres-ponds to the size of the expected fusion protein and whichreacts ~ith antibodies against insulin. After the fusion protein has been isolated the expected proinsulin derivative can be liberated by cleavage with cyanogen bromide. After disruption of the bacteria (French Press;
(R)Dyno-mill ) and centrifugation, the L', D'-proinsu-lin fusion protein is located in the sediment, so that con-siderable amounts of the other proteins can nou be removed ~ith the supernatant.
The stated induction conditions apply to shake cultures;
for Larger fermentations it is advantageous to modify the OD
values accordingly and, ~here appropriate, to vary the IPTG
concentrations slightly.
Example 7 An overnight culture in LB medium containing SO ~g/ml ampi-cillin is prepared from E. co~i 294 transformants which contain the plasmid pH154/2S (Figure 3) and the next morn-ing ;s diluted in the ratio of about 1:100 in M9 medium (J. H. Miller, Loc. cit.) containing 2000 ~g/ml casamino acids and 1 ~g/ml thiamine. ~hen the OD = O.S indolyl-3-acryl;c acid is added so that the final concentration is15 ~g/ml. After incubation for 2 to 3 hours, the bacteria are removed by centr;fugation. SDS gel electrophoresis shows a very pronounced protein band ~hich is at the place ex-pected for the fus;on protein and which reacts ~ith anti-bodies against insulin. After disruption of the bacteriaand centrifugation, the L', D'-proinsu~in fusion protein - ~ - 15 -is located in the sediment so that, again, considerable amounts of the other proteins are no~ removed with the supernatant.
In the present case too, the stated induction conditions apply to shake cultures. Fermentations in larger volumes require altered concentrations of casamino acids or addi-tion of L-tryptophan.
Example 8 The plasmid pH154/25 (Figure 3) is opened ~ith EcoRI, and the protruding DNA single strands are filled in ~ith Klenow polymerase. The DNA thus obtained is incubated ~ith the enzyme MluI, and the DNA coding for insulin is cut out of the plasmid. Separation by gel electrophoresis is carried out to remove this fragment from the remainder of the plas-mid, and the remainder of the plasmid is isoLated.
The plasmid sho~n in Figure 3 of German Offenlegungsschrift3,429,430 (European Patent Application A1 0,171,024) is reacted with the restriction enzymes AccI and SaLI, and the DNA fragment containing the hirudin sequence is removed.
After the protruding ends of the SalI restriction site have been filled in with Klenow polymerase, the DNA segment is ligated ~ith the synthetic DNA of the formula (N8) Met Thr 5' CCC ACG CGT ATG ACG T 3' 3' GGG TGC GCA TAC TGC ATA 5' (N8) The ligation product is incubated with MluI. After the en-zyme has been inactivated at 65C, the DNA mixture is treated ~ith bovine alkaline phosphatase at 37C for one hour.
This is followed by removal of the phosphatase and the re-striction enzyme from the mixture by extraction with phenol,and the DNA is purified by ethanol precipitation. The DNA
~hich has thus been treated is inserted using T4 ligase -~ 1 336329 ;nto the opened remainder of the plasmid pH154/25, this resulting in the plasmid pK150 which has been characterized by restriction anaLysis and DNA sequencing by the method of Maxam and Gilbert (Figure 7).
Example 9 E. coli 29~ bacter;a which contain the plasmid pK150 (Fi-gure 7) are cultured in LB medium containing 30 to 50 ~g/ml ampicillin at 37C overnight. The culture is diLuted in the ratio of 1:100 ~ith M9 medium which contains 2000 ~g/ml casamino acids and 1 ~g/ml thiamine, and the mixture is incubated at 37C, mixing continuously. ~hen the OD600= 0.5 or 1, indolyl-3-acrylic acid is added to a final concentration of 15 ~/ml, and the mixture is incubated for 2 to 3 hours or 16 hours respect;vely. The bacteria are then removed by centrifugation and disrupted in 0.1 M
sodium phosphate buffer tpH 6.5) under pressure. The spar-ingly soluble proteins are removed by centrifugation and analyzed by SDS polyacryLamide gel electrophoresis. It emerges that cells whose trp operon has been induced con-tain in the region below 20,000 Daltons but above14,000 Da~-tons a new protein ~hich is not found in non-induced cells.
After the fusion protein has been isolated and reacted with cyanogen bromide hirudin is liberated.
Example 10 The constructs described hereinafter permit the introduc-tion, upstream of the 5'-end of the trp-D sequence, of DNA
sequences which contain as man~ recognition sites for various restriction enzymes as possible in order to incorporate the trp-D sequence into as many as possible of the wide variety of prokaryoctic expression systems.
The plasmids pUC12 and pUC13 (Pharmacia P-L Biochemicals, 5401 St. Goar: The Molecular Biology Catalogue 1983, Ap-~ - 17 - 1 336329 pendix, p. 89) contain ~ polylinker se~uence, it being the intention to ;nsert in the pl~sm;d pUC13, bet~een the restric-tion cleavage sites for XmaI ~nd SacI, the Mstl-HindIII trp fr~gment from the pl~smid p106/4 (Figure 2) ~hich is fused vith the HindIII-hirudin-SacI fragment from the plasmid pK150 ~Figure 7).
For this purpose, the DNA of the pl~snid pUC13 is first treated ~ith the restriction enzyme XmaI. The ends of the ~inearized plasmid are fil~ed in by ~eans of the Kleno~
polymerase reaction. After ethanol precipitation, the DNA
is treated ~ith the enzy~e SacI and is ~g~in precipitated fro0 the reaction mixture ~ith ethanol. The DNA is no~
reacted in ~n aqueous ligation ~ixture Yith the MstI-HindIlI trp-D fragment isol~ted from plasmid pH106/4 and ~ith the HindIII-Sac~ hirudin fra~ment isolated from the plas~id pK150, ~nd T4 DNA ligase.
The plasmid pK160 thus obtained no~ cont~ins, i~mediately upstream of the trp-D sequence, aultiple restriction en-zyme recognition sequence ~hich e~br~ces restriction sites for the enz~nes XmaI, S~aI, BamHI, XbaI, HincII, SalI, AccI, PstI ~nd HindIII. Furthermore, ~n EcoRI restriction site is generated do~nstre-m of the 3'-end of the hirudin se-~uence in this construction (Figure 8).
Example 11 The plasmid pH131/5 is prepared as follows:
The plas~id ptrpL1 is opened with ClaI and ligated with the synthe-tically prepared, self-comPlement~ry oligonucleotide (N9) 5' pCGACCATGGT 3'; ~N9) .
~he plasmid pH131/5 (Figure 9) thus obtained is opened at the restriction site, vhich has been introduced in this 0anner, - ~ ~ 336329 _ - 18 -~ for the restriction enzyme NcoI, and the resulting pro-truding single-stranded ends are filled in by means of the - KLenov polymerase reaction. The linearized, blunt-ended DNA is now cut w;th the enzyme EcoRI, and the lar~er of the two resuLting DNA sequences is separated from the smal-ler sequence by ethanol precipitation. The rema;nder of the pLasm;d DNA of the plasmid pH131/5 thus obtained is now Ligated with a fragment from pK160 coding for trp-D'-hirudin by reaction vith T4 ligase. This fragment is cleaved out of the p~asm;d pK160 by opening the plasm;d w;th HincII and EcoRI. The fragment is removed from the remainder of the plasmid by gel eLectrophoresis, and ;s then eluted from the gel material. The Ligation product ;s transformed ;nto E. coLi K12. The clones containing plasmid DNA are ;solated and characterized by restriction anaLysis and DNA sequence analysis. The plasmid pK170 thus obtained contains fused onto the trp operator a DNA
sequence which codes for Met-Asp-Ser-Arg-GLy-Ser-Pro-Gly-trp-D'-(hirudin~ (Figure 9).
ExampLe 12 The pLasm;d pJF118 (Example 6) is opened with EcoRI, and the protruding DNA ends are converted into bLunt ends by means of the KLenow polymerase reaction. The DNA thus treated is then cut with the enzyme SalI, and the short EcoRI-SaLI fragment is removed by gel eLectrophoresis.
The pLasmid pK 170 (Exsmple 11) is cleaved with NcoI, and the protruding ends are converted into bLunt ends using Klenov polymerase. The plasmid DNA is removed from the reaction mixture by ethanol precipitat;on and ;s treated with the enzymes HindIII and BamHI. Two of the resuLting fragments are ;solated, namely the NcoI (w;th fiLled-in end)-trp-D'-HindIII fragment and the H;ndIII-h;rud;n-~amHI
fragment (German Offenlegungsschr;ft 3,429,430). The two fragments are isolated after separation by geL eLectro-phoresis.
-- 1 33632~
In addition, the BamHI-SalI-HirudinII fragment shown in Figure 2 of German Offenlegungsschrift 3,429,430 is iso-lated. In a ligation reaction the four fragments, namely the remainder of the plasmid pJF 118, the NcoI-trp-D'-HindIII fragment, the HindIII-hirudin-BamHI fragment and the hirudinII fragment, are now reacted together and the resuLting plasmid pK 180 (Figure 10) is transformed into E. col; K12-W 3110. Correct plasmids are shown by it be;ng possible to detect an EcoRI-trp-D'-hirudin-SalI fragment in the pLasmid DNA. The trp-D'-hirudin sequence is now attached to the tac promoter. The fusion protein is ex-pressed as in Example 6.
Example 13 The plasmids derived from pBR 322, such as pH120/14 (Ex-ample 1, Figure 1), pH154/25 (ExampLe 1, Figure 3), pH256 (Example 4, Figure 5), pK150 (Example 8, Figure 7) and pK170 (Example 11, Figure 9), have - on the figures in the clockwise direction - between the start codon of the fusion protein and the next HindIII site (corresponding to HindIII
at position 29 in pBR322) an additional PvuII site ;n the region of the fragment which contains the trp promoter and operator, but outside the promoter region.
It has now been found that by deletion of the DNA segment which is bounded by the PvuII site described and the PvuII
site which corresponds to position 2066 in pBR322, the yield of a cloned protein (or fusion protein) is dis~inctly in-creased~
The shortening of the p~asmid pH154/25 to give pH154/25*
is described by way of example hereinafter, it being pos-s;ble for this to be effected correspondingly for the other plasmids mentioned above (the shortened plasmids likewise being identified by 3n asterisk):
pH154/25 is reacted with PvulI ~in accordance with the manu--1 33632~
facturer's instructions), resulting ;n three fragments:
t Fragment 1: From the PvuII restriction site of the proin-sulin gene to the PvuII restriction site cor-respond;ng to position 2066 in pBR322, Fragment 2: from the PvuII restriction site near the trp promoter to the PvuII site of the proinsulin gene and Fragment 3: from the PvuII site near the trp promoter fragment to the PvuII site corresponding to position 2066 in pBR322.
The fragments can be separated by electrophoresis on agarose and then isolated (Maniatis eb al., Molecular Cloning, Cold Spring Harbor, 1982).
The fragments 1 and 2 are joined under blunt end conditions using the enzyme T4 DNA ligase. Transformation into E. coli 294 is followed by testing for those colonies ~hich contain a plasmid ~ith the complete proinsulin sequence and thus have the fragments in the desired order. The pLasmid pH154/25* is depicted in Figure 11.
A distinct increase in the proportion of fusion protein is observed on expression, ~hich is carried out as described in the preceding examples.
Example 14 The plasmid pH 154/25* ~Example 13, Figure 11) is digested ~ith HindIII and SalI, and the small fragment (having the proinsulin sequence) is separated off by gel eLectrophore-sis. The large fragment is isolated and ligated ~ith the synthetic DNA (N10) (Ala) 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
The plasmid pInt13 (Figure 12) is produced.
The DNA (N10) codes for an amino acid sequence which con-tains several cleavage sites for chemical cleavage:
a) Met for cyanogen bro-ide, b) Trp for N-bromosuccinimide (NBS or BSI) c) Asp-Pro for proteolytic cleavage, the upstream Glu ad-ditionally weakening the Asp-Pro bond to~ards the action of acids.
The introduction of this HindlII-Sa~l-linker (N10) into the reading frame of a coded polypeptide thus allo~s the options which have been mentioned for chemical cleavage off, de-pending on the amino acid sequence of the desired prote;n and on its sensitivity to the cleaving agents.
The figures are not to scale~
' ~ - 22 - 1 336329 Amino ac;d sequence I
23) Ser Asn Gly His Asn Val Val Ile Tyr Arg Asn His Ile Pro Ala Gln Thr Leu Ile Glu Arg Leù A~a Thr Met Ser Asn Pro Val Leu Met Leu Ser Pro Gly Pro Gly VaL Pro Ser Glu Ala Gly Cys Met Pro Glu Leu Leu Thr Arg Leu Arg Gly Lys Leu Pro ILe Ile Gly ILe Cys Leu Gly (93) His Gln ALa Ile Val Glu ALa
Claims (20)
1. A fusion protein of the 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 representing the desired final protein.
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 representing the desired final protein.
2. A fusion protein as claimed in claim 1, wherein n is one and X comprises 1 to 5 amino acids.
3. A fusion protein as claimed in claim 1, wherein n is one and Lys-Ala is located at the N-terminal end of X.
4. A fusion protein as claimed in claim 1, 2 or 3, wherein Y contains at the C-terminal end Met, Cys, Trp, Arg or Lys or one of the groups (Asp)m-Pro or Glu-(Asp)m-Pro or Ile-Glu-Gly-Arg, in which m de-notes 1, 2 or 3, or consists of these amino acids or groups.
5. A fusion protein as claimed in claim 1, 2 or 3, wherein Z denotes the amino acid sequence of human proinsulin or of a hirudin.
6. A process for the preparation of the fusion proteins as defined in claim 1, wherein a gene structure coding for these fusion proteins is expressed in a host cell, and the fusion protein is separated off.
7. The process as claimed in claim 6, wherein the DNA
sequence I
DNA Sequence I
codes in the gene structure for D'.
sequence I
DNA Sequence I
codes in the gene structure for D'.
8. The process as claimed in claim 6, wherein the DNA sequence (coding strand) 5' AAA GCA AAG GGC 3' codes in the gene structure for X.
9. The process as claimed in claim 6, wherein the gene structure is selected so that the fusion protein is insoluble.
10. The process as claimed in claim 6, wherein the gene structure is contained in phase in a vector which contains the promoter, the operator and the ribosome binding site of the L-peptide from the trp operon of E. coli.
11. The process as claimed in claim 10, wherein the vector is a derivative of pBR322, the segment from the HindIII site at position 29 to the PvuII site at position 2066 having been deleted from the pBR322 DNA.
12. The process as claimed in claim 6, wherein the host cell is a bacterium.
13. The process as claimed in claim 6, wherein the host cell is E. coli.
14. A gene structure coding for fusion proteins as claimed in claim 1.
15. A vector containing a gene structure as claimed in claim 14.
16. A derivative of the plasmid pBR322, the segment from the HindIII site at position 29 to the PvuII site at position 2066 having been deleted from the pBR322 DNA, con-taining a gene structure as claimed in claim 14.
17. An expression system containing a vector as claimed in claim 15 or 16.
18. E. coli containing a vector as claimed in claim 15 or 16.
19. A process for the preparation of a eukaryotic pro-tein, which comprises cleavage off, chemically or enzymati-cally, of the amino acid sequence Z from a fusion protein as claimed in claim 1, 2 or 3.
20. Plasmids pH 154/25, pH 254, pH 255, pH 256, pH257, pH 120/14, pK 150, pK 160, pK 170, pK 180, pH 154/25*, pH 256*, pH 120/14*, pK 150*, pK 170* and pInt13.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19853526995 DE3526995A1 (en) | 1985-07-27 | 1985-07-27 | FUSION PROTEINS, METHOD FOR THEIR PRODUCTION AND THEIR USE |
| DEP3526995.2 | 1985-07-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1336329C true CA1336329C (en) | 1995-07-18 |
Family
ID=6276985
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|---|---|---|---|
| CA000514682A Expired - Lifetime CA1336329C (en) | 1985-07-27 | 1986-07-25 | Fusion proteins, a process for their preparation and their use |
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|---|---|
| EP (1) | EP0211299B1 (en) |
| JP (1) | JPH07113040B2 (en) |
| KR (1) | KR950000299B1 (en) |
| AT (1) | ATE50596T1 (en) |
| AU (1) | AU595221B2 (en) |
| CA (1) | CA1336329C (en) |
| DE (2) | DE3526995A1 (en) |
| DK (1) | DK172618B1 (en) |
| ES (1) | ES2000278A6 (en) |
| FI (1) | FI86887C (en) |
| GR (1) | GR861957B (en) |
| HU (1) | HU197356B (en) |
| IE (1) | IE59127B1 (en) |
| IL (1) | IL79522A (en) |
| NO (1) | NO175640C (en) |
| NZ (1) | NZ216967A (en) |
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| US7476652B2 (en) | 2002-06-18 | 2009-01-13 | Sanofi-Aventis Deutschland Gmbh | Acidic insulin preparations having improved stability |
| US9364519B2 (en) | 2011-09-01 | 2016-06-14 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition for use in the treatment of a neurodegenerative disease |
| US9408893B2 (en) | 2011-08-29 | 2016-08-09 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical combination for use in glycemic control in diabetes type 2 patients |
| US9526764B2 (en) | 2008-10-17 | 2016-12-27 | Sanofi-Aventis Deutschland Gmbh | Combination of an insulin and a GLP-1-agonist |
| US9707176B2 (en) | 2009-11-13 | 2017-07-18 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition comprising a GLP-1 agonist and methionine |
| US9950039B2 (en) | 2014-12-12 | 2018-04-24 | Sanofi-Aventis Deutschland Gmbh | Insulin glargine/lixisenatide fixed ratio formulation |
| US9981013B2 (en) | 2010-08-30 | 2018-05-29 | Sanofi-Aventis Deutschland Gmbh | Use of AVE0010 for the treatment of diabetes mellitus type 2 |
| US10029011B2 (en) | 2009-11-13 | 2018-07-24 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition comprising a GLP-1 agonist, an insulin and methionine |
| US10159713B2 (en) | 2015-03-18 | 2018-12-25 | Sanofi-Aventis Deutschland Gmbh | Treatment of type 2 diabetes mellitus patients |
| US10434147B2 (en) | 2015-03-13 | 2019-10-08 | Sanofi-Aventis Deutschland Gmbh | Treatment type 2 diabetes mellitus patients |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3738541A1 (en) * | 1987-11-13 | 1989-05-24 | Hoechst Ag | METHOD FOR INSULATING AND CLEANING HIRUDINE |
| DE3541856A1 (en) * | 1985-11-27 | 1987-06-04 | Hoechst Ag | EUKARYOTIC FUSION PROTEINS, THEIR PRODUCTION AND USE, AND MEANS FOR CARRYING OUT THE PROCESS |
| ES2035043T3 (en) * | 1986-03-12 | 1993-04-16 | Imcera Group Inc. | HYBRID EXPRESSION VECTORS, THEIR CONSTRUCTION AND USES. |
| JPH03503596A (en) * | 1987-06-24 | 1991-08-15 | ノボ・ノルディスク エー／エス | A method for preparing a protein or polypeptide, a DNA sequence encoding the polypeptide, a microorganism having the DNA sequence and the polypeptide, and use of the polypeptide as a pharmaceutical preparation |
| US5179196A (en) * | 1989-05-04 | 1993-01-12 | Sri International | Purification of proteins employing ctap-iii fusions |
| IL95495A (en) * | 1989-08-29 | 1996-10-16 | Hoechst Ag | Fusion proteins their preparation and use |
| US5227293A (en) * | 1989-08-29 | 1993-07-13 | The General Hospital Corporation | Fusion proteins, their preparation and use |
| US5358857A (en) * | 1989-08-29 | 1994-10-25 | The General Hospital Corp. | Method of preparing fusion proteins |
| GB8927722D0 (en) * | 1989-12-07 | 1990-02-07 | British Bio Technology | Proteins and nucleic acids |
| DE3942580A1 (en) * | 1989-12-22 | 1991-06-27 | Basf Ag | METHOD FOR PRODUCING HIRUDINE |
| ATE264871T1 (en) | 1996-07-26 | 2004-05-15 | Aventis Pharma Gmbh | INSULIN DERIVATIVES WITH INCREASED ZINC BINDING |
| DE19726167B4 (en) | 1997-06-20 | 2008-01-24 | Sanofi-Aventis Deutschland Gmbh | Insulin, process for its preparation and pharmaceutical preparation containing it |
| DE19825447A1 (en) | 1998-06-06 | 1999-12-09 | Hoechst Marion Roussel De Gmbh | New insulin analogues with increased zinc formation |
| DE10114178A1 (en) | 2001-03-23 | 2002-10-10 | Aventis Pharma Gmbh | Zinc-free and low-zinc insulin preparations with improved stability |
| JP5368450B2 (en) | 2007-09-21 | 2013-12-18 | カディラ・ヘルスケア・リミテッド | Fusion protein system suitable for high expression of peptides |
| US9821032B2 (en) | 2011-05-13 | 2017-11-21 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical combination for improving glycemic control as add-on therapy to basal insulin |
| KR20200080747A (en) | 2018-12-27 | 2020-07-07 | 주식회사 폴루스 | An Enzymatic Conversion Composition for Producing Insulin from Proinsulin and a Method for Producing Insulin from Proinsulin Using the Same |
| KR20200080748A (en) | 2018-12-27 | 2020-07-07 | 주식회사 폴루스 | A Method for Purifying Proinsulin Using Anion Exchange Chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA802992B (en) * | 1979-06-01 | 1981-10-28 | Univ California | Human pre-growth hormone |
| ZA811368B (en) * | 1980-03-24 | 1982-04-28 | Genentech Inc | Bacterial polypedtide expression employing tryptophan promoter-operator |
| WO1986005513A1 (en) * | 1985-03-18 | 1986-09-25 | Gene Labs, Inc. | Hybrid-gene cassette vector |
| GB8507666D0 (en) * | 1985-03-25 | 1985-05-01 | Wellcome Found | Epidermal growth factor production |
| DE3650011T2 (en) * | 1985-04-08 | 1994-11-17 | Genetic Systems Corp | EXPRESSION AND DIAGNOSIS WITH GAG-CODED PEPTIDES THAT ARE IMMUNOLOGICALLY REACTIVE WITH ANTIBODIES AGAINST LAV. |
-
1985
- 1985-07-27 DE DE19853526995 patent/DE3526995A1/en not_active Withdrawn
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1986
- 1986-07-19 AT AT86109945T patent/ATE50596T1/en active
- 1986-07-19 DE DE8686109945T patent/DE3669175D1/en not_active Expired - Lifetime
- 1986-07-19 EP EP86109945A patent/EP0211299B1/en not_active Expired - Lifetime
- 1986-07-24 FI FI863041A patent/FI86887C/en not_active IP Right Cessation
- 1986-07-24 ES ES8600544A patent/ES2000278A6/en not_active Expired
- 1986-07-25 PT PT83065A patent/PT83065B/en unknown
- 1986-07-25 GR GR861957A patent/GR861957B/en unknown
- 1986-07-25 AU AU60565/86A patent/AU595221B2/en not_active Expired
- 1986-07-25 DK DK198603554A patent/DK172618B1/en not_active IP Right Cessation
- 1986-07-25 NO NO863000A patent/NO175640C/en unknown
- 1986-07-25 IL IL79522A patent/IL79522A/en not_active IP Right Cessation
- 1986-07-25 ZA ZA865556A patent/ZA865556B/en unknown
- 1986-07-25 IE IE197786A patent/IE59127B1/en not_active IP Right Cessation
- 1986-07-25 CA CA000514682A patent/CA1336329C/en not_active Expired - Lifetime
- 1986-07-25 HU HU863100A patent/HU197356B/en unknown
- 1986-07-25 NZ NZ216967A patent/NZ216967A/en unknown
- 1986-07-26 JP JP61176558A patent/JPH07113040B2/en not_active Expired - Lifetime
- 1986-07-26 KR KR1019860006142A patent/KR950000299B1/en not_active IP Right Cessation
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7713930B2 (en) | 2002-06-18 | 2010-05-11 | Sanofi-Aventis Deutschland Gmbh | Acidic insulin preparations having improved stability |
| US7476652B2 (en) | 2002-06-18 | 2009-01-13 | Sanofi-Aventis Deutschland Gmbh | Acidic insulin preparations having improved stability |
| US10117909B2 (en) | 2008-10-17 | 2018-11-06 | Sanofi-Aventis Deutschland Gmbh | Combination of an insulin and a GLP-1 agonist |
| US9526764B2 (en) | 2008-10-17 | 2016-12-27 | Sanofi-Aventis Deutschland Gmbh | Combination of an insulin and a GLP-1-agonist |
| US10029011B2 (en) | 2009-11-13 | 2018-07-24 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition comprising a GLP-1 agonist, an insulin and methionine |
| US9707176B2 (en) | 2009-11-13 | 2017-07-18 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition comprising a GLP-1 agonist and methionine |
| US10028910B2 (en) | 2009-11-13 | 2018-07-24 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition comprising a GLP-1-agonist and methionine |
| US9981013B2 (en) | 2010-08-30 | 2018-05-29 | Sanofi-Aventis Deutschland Gmbh | Use of AVE0010 for the treatment of diabetes mellitus type 2 |
| US9408893B2 (en) | 2011-08-29 | 2016-08-09 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical combination for use in glycemic control in diabetes type 2 patients |
| US9987332B2 (en) | 2011-09-01 | 2018-06-05 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition for use in the treatment of a neurodegenerative disease |
| US9364519B2 (en) | 2011-09-01 | 2016-06-14 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition for use in the treatment of a neurodegenerative disease |
| US9950039B2 (en) | 2014-12-12 | 2018-04-24 | Sanofi-Aventis Deutschland Gmbh | Insulin glargine/lixisenatide fixed ratio formulation |
| US10434147B2 (en) | 2015-03-13 | 2019-10-08 | Sanofi-Aventis Deutschland Gmbh | Treatment type 2 diabetes mellitus patients |
| US10159713B2 (en) | 2015-03-18 | 2018-12-25 | Sanofi-Aventis Deutschland Gmbh | Treatment of type 2 diabetes mellitus patients |
Also Published As
| Publication number | Publication date |
|---|---|
| NZ216967A (en) | 1988-08-30 |
| JPH07113040B2 (en) | 1995-12-06 |
| FI86887B (en) | 1992-07-15 |
| NO863000D0 (en) | 1986-07-25 |
| KR950000299B1 (en) | 1995-01-13 |
| FI863041A0 (en) | 1986-07-24 |
| PT83065B (en) | 1989-02-28 |
| NO175640B (en) | 1994-08-01 |
| DK355486A (en) | 1987-01-28 |
| ZA865556B (en) | 1987-03-25 |
| DE3669175D1 (en) | 1990-04-05 |
| ATE50596T1 (en) | 1990-03-15 |
| JPS6229600A (en) | 1987-02-07 |
| DK355486D0 (en) | 1986-07-25 |
| DE3526995A1 (en) | 1987-02-05 |
| FI863041A (en) | 1987-01-28 |
| AU6056586A (en) | 1987-01-29 |
| DK172618B1 (en) | 1999-03-01 |
| IL79522A0 (en) | 1986-10-31 |
| IE59127B1 (en) | 1994-01-12 |
| NO175640C (en) | 1994-11-09 |
| EP0211299A2 (en) | 1987-02-25 |
| EP0211299A3 (en) | 1988-06-01 |
| EP0211299B1 (en) | 1990-02-28 |
| ES2000278A6 (en) | 1988-02-01 |
| AU595221B2 (en) | 1990-03-29 |
| HUT43629A (en) | 1987-11-30 |
| IE861977L (en) | 1987-01-27 |
| PT83065A (en) | 1986-08-01 |
| FI86887C (en) | 1992-10-26 |
| IL79522A (en) | 1991-08-16 |
| GR861957B (en) | 1986-11-25 |
| KR870001312A (en) | 1987-03-13 |
| NO863000L (en) | 1987-01-28 |
| HU197356B (en) | 1989-03-28 |
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