CA1338338C - Process for the preparation of foreign proteins in streptomycetes - Google Patents
Process for the preparation of foreign proteins in streptomycetesInfo
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- CA1338338C CA1338338C CA000565869A CA565869A CA1338338C CA 1338338 C CA1338338 C CA 1338338C CA 000565869 A CA000565869 A CA 000565869A CA 565869 A CA565869 A CA 565869A CA 1338338 C CA1338338 C CA 1338338C
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- Prior art keywords
- tendamistat
- gene
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- streptomycetes
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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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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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
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- Proteomics, Peptides & Aminoacids (AREA)
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- Diabetes (AREA)
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- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
Abstract
The tendamistat gene can be used for the construction of fused genes with which fusion proteins are expressed and excreted in Streptomycetes host cells. The tendamistat portion can be modified, in particular it can be C-terminal shortened.
Description
A process for the preparation of foreign proteins in Streptomycetes European Patent Application 85 105 610.1 (Filed May 8, 1985, published November 21, 1985 as No. 0 161 629, Applicant Hoechst Aktiengesellschaft), and South African Patent 85/3672 (issued December 24, 1985) disclose the use of the DNA coding for the signal peptide (prepeptide) of the a-amylase inhibitor tendamistat in order for a Streptomycetes cell to excrete a polypeptide, in particular tendamistat. The appropriate DNA can, in principle, be obtained from every strain producing tendamistat, but a DNA obtained as in Example 3 of German Offenlegungsschrift 3,331,860 (published March 21, 1985, Applicant Hoechst Aktiengesellschaft) is preferably used.
Canadian Patent Application 560,579 (Filed March 4, 1988) has already proposed a process for the excretion of fusion proteins from Streptomycetes, which comprises incorporating the coding sequence has been modified where appropriate, and expressing the recombinant gene in a Streptomycetes cell. Thus, in this case the tendamistat structural gene is used as a "carrier" for another gene, the fusion proteins which are obtained having the amino acid sequence of another protein located within the tendamistat amino acid sequence.
Consequently, on chemical or enzymatic cleavage of this fusion protein to liberate the other protein, two tendamistat part-sequences are obtained. The said earlier application also relates to tendamistat derivatives, which are understood to include those with a markedly shortened amino acid chain. Derivatives of this type are able in a reversible manner to react with the specific receptors in the form of a competitive inhibitory mechanism.
It has now been found that foreign proteins can also be prepared in Streptomycetes by constructing fusion protein ~ - 2 - 1338338 genes in whi ch the structural gene for t:he desired protein is ooupled to the 3' end (of the coding strand) of the t~n~m;.~-tat gene, ~hich has been modified where appropriate. The modification of the tendamistat gene may comprise, in S particular, C-terminal shortening.
The DNA coding for tendamistat is depicted in EP-A
0,161,629 (where it is DNA sequence C; Table 1 in the annex). This structural gene contains several cleavage sites for restriction enzymes, which can be used to modi-fy the coded amino acid sequence. Suitable cleavage sites are those for BstEII in the region of triplets 31 and 32, StuI in the region of triplets 43 and 4~, and Sau3A in the region of triplets 52 and 53. It is possible, by in-corporation of appropriate linkers, to insert at these sites one or more additional amino acids, to eliminate DNA segments between these cleavage sites, or to code for shortened amino acid sequences by incorporation of stop codons. Furthermore, it is possible by site-specific mutagenesis for any desired amino acids to be inserted, replaced or eliminated. In this way proteins are obtained which have an ~-amylase inhibitory action, as well as proteins which do not have this activity but still react with the corresponding receptors.
O The invention also relates to appropriate gene structures, vectors containing these gene structures, Streptomycetes cells transformed with these vectors, the excreted fusion proteins, and their use for the preparation of the foreign proteins and tendamistat derivatives. Preferred embodi-ments of the invention are explained in detail herein-after and defined in the patent claims.
figures 1 to ~ depict some plasmid constructions accord-ing to the invention.
Fig. 1 shows the preparation of the hybrid plasmid pKK310 which codes for a fusion protein in which part of the tendamistat amino acid sequence is follo~ed by a bridging member of seven amino acids and, thereafter, the amino acid sequence of monkey proinsulin.
Fig. 2 sho~s the construction of the expression plasmid pTF1 starting from the plasmid pKK310.
Fig. 3 sho~s the construction of the plasmid pRS10 in ~hich part of the tendamistat gene is followed by the polylinker from pUC18, and its reconstruction into the expression plasmid pTF10. "mcs" denotes the polylinker region (multiple cloning site) of pUC18.
Finally, Fig. 4 sho~s the construction of the plasmid 15 pKK400 ~hich codes for a fusion protein in which the ~hole of the amino acid sequence of tendamistat is follo~ed by a bridging member of eleven amino acids and, thereafter, ~ the amino acid sequence of monkey proinsulin, and its reconstruction into the expression plasmid pGF1.
The figures are not dra~n true to scale.
The fusion proteins obtained according to the invention, ~hich are exported from the cell, have the advantage that 25 they can readily be isolated from the culture filtrate.
O The isolation can be carried out in a manner known per se, advantageously by adsorption or ion exchange chroma-tography and/or gel filtration.
30 The desired foreign protein (fusion partner) is liberated by enzymatic or chemical cleavage like~ise in a manner known per se.
In this connection, the type of cleavage depends, in par-35 ticular, on the amino acid sequence of the desired pro-tein. It will be expedient in many cases to incorporate a connecting member or bridging member in the cleavage site bet~een the tendamistat sequence and the amino acid ` _ 4 _ I 33 83 3~
~
sequence of the desired protein. If the desired protein contains, for example, no methionine, the connecting mem-ber can denote Met, whereupon chemical cleavage with cyanogen chloride or bromide is carried out. If the con-necting member has a carboxyl-terminal cysteine, or if the connecting member represents Cys, it is possible for enzymatic cysteine-specific cleavage, or chemical cleav-age, for example after specific S-cyanylation, to follow.
If the bridging member has a carboxyl-terminaL tryptophan, or if the connecting member represents Trp, chemical cleavage with N-bromosuccinimide can be carried out.
Desired proteins which do not contain Asp-Pro in their a0ino acid sequence and are sufficiently stable to acid can, as fusion proteins having this bridging member, be cleaved proteolytically in a manner known per se. This results in proteins which contain N-terminal prolineand C-terminal aspartic acid. Thus, it is also possible in this way to synthesize modified proteins.
The Asp-Pro bond can be made even more labile to acid if this bridging member denotes (Asp)n-Pro or is Glu-~Asp)n-Pro, n denoting 1 to 3.
Examples of enzymatic cleavages have also been disclosed, O it also being possible to use modified enzymes having ioproved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, it is expedient to choose a peptide sequence in which an amino acid which can be eliminated by trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of the proinsulin, for example Ala-Ser-Met-Thr-Arg, since it is then possible to carry out the arginine-specific cleavage using the protease trypsin.
If the desired protein does not contain the amino acid sequence - _ 5 _ 1338338 Ile-Glu-Gly-Arg, the fusion protein having the corresponding bridging member can be cleaved with factor Xa (European Patent Application published under No. EP-A 0,025,190 on March 18, 1981 and European Patent Application published under No. EP-A 0,161,973 on November 21, 1985.
The isolation of the cleavage products depends on the properties of these proteins. Concerning the isolation of tendamistat and its derivatives, reference may be made to the literature cited in German Offenlegungsschrift 3,331,860 (published March 21, 1985, Applicant Hoechst Aktiengesellschaft).
The invention is explained in detail in the examples which follow.
Unless other~ise indicated, percentage data relate to weight. The figures having the same numbers as the examples relate thereto.
Ex~mple 1 The starting material used is the plasmid pKAI650, ~hich is described in European Patent Application 86 113 627.3 (Filed October 2, 1986, published April 15, 1987 as No. 0 218 204, Applicant Hoechst Aktiengesellschaft). This plasmid can be obtained from the plasmid pKAIl, which is described in German Offenlegungsschrift 3,331,860 (published March 21, 1985, Applicant Hoechst Aktiengesellschaft), by isolation of the 650 bp HincII/SstI fragment and cloning into the plasmid pUCl9 which has been opened with these enzymes. The unique HindIII cleavage site in this plasmid is removed (by cutting with this enzyme, filling in the protruding ends, and ligation) to result in the plasmid pKAI650a (1).
Canadian Patent Application 560,579 (Filed March 4, 1988) has already proposed a process for the excretion of fusion proteins from Streptomycetes, which comprises incorporating the coding sequence has been modified where appropriate, and expressing the recombinant gene in a Streptomycetes cell. Thus, in this case the tendamistat structural gene is used as a "carrier" for another gene, the fusion proteins which are obtained having the amino acid sequence of another protein located within the tendamistat amino acid sequence.
Consequently, on chemical or enzymatic cleavage of this fusion protein to liberate the other protein, two tendamistat part-sequences are obtained. The said earlier application also relates to tendamistat derivatives, which are understood to include those with a markedly shortened amino acid chain. Derivatives of this type are able in a reversible manner to react with the specific receptors in the form of a competitive inhibitory mechanism.
It has now been found that foreign proteins can also be prepared in Streptomycetes by constructing fusion protein ~ - 2 - 1338338 genes in whi ch the structural gene for t:he desired protein is ooupled to the 3' end (of the coding strand) of the t~n~m;.~-tat gene, ~hich has been modified where appropriate. The modification of the tendamistat gene may comprise, in S particular, C-terminal shortening.
The DNA coding for tendamistat is depicted in EP-A
0,161,629 (where it is DNA sequence C; Table 1 in the annex). This structural gene contains several cleavage sites for restriction enzymes, which can be used to modi-fy the coded amino acid sequence. Suitable cleavage sites are those for BstEII in the region of triplets 31 and 32, StuI in the region of triplets 43 and 4~, and Sau3A in the region of triplets 52 and 53. It is possible, by in-corporation of appropriate linkers, to insert at these sites one or more additional amino acids, to eliminate DNA segments between these cleavage sites, or to code for shortened amino acid sequences by incorporation of stop codons. Furthermore, it is possible by site-specific mutagenesis for any desired amino acids to be inserted, replaced or eliminated. In this way proteins are obtained which have an ~-amylase inhibitory action, as well as proteins which do not have this activity but still react with the corresponding receptors.
O The invention also relates to appropriate gene structures, vectors containing these gene structures, Streptomycetes cells transformed with these vectors, the excreted fusion proteins, and their use for the preparation of the foreign proteins and tendamistat derivatives. Preferred embodi-ments of the invention are explained in detail herein-after and defined in the patent claims.
figures 1 to ~ depict some plasmid constructions accord-ing to the invention.
Fig. 1 shows the preparation of the hybrid plasmid pKK310 which codes for a fusion protein in which part of the tendamistat amino acid sequence is follo~ed by a bridging member of seven amino acids and, thereafter, the amino acid sequence of monkey proinsulin.
Fig. 2 sho~s the construction of the expression plasmid pTF1 starting from the plasmid pKK310.
Fig. 3 sho~s the construction of the plasmid pRS10 in ~hich part of the tendamistat gene is followed by the polylinker from pUC18, and its reconstruction into the expression plasmid pTF10. "mcs" denotes the polylinker region (multiple cloning site) of pUC18.
Finally, Fig. 4 sho~s the construction of the plasmid 15 pKK400 ~hich codes for a fusion protein in which the ~hole of the amino acid sequence of tendamistat is follo~ed by a bridging member of eleven amino acids and, thereafter, ~ the amino acid sequence of monkey proinsulin, and its reconstruction into the expression plasmid pGF1.
The figures are not dra~n true to scale.
The fusion proteins obtained according to the invention, ~hich are exported from the cell, have the advantage that 25 they can readily be isolated from the culture filtrate.
O The isolation can be carried out in a manner known per se, advantageously by adsorption or ion exchange chroma-tography and/or gel filtration.
30 The desired foreign protein (fusion partner) is liberated by enzymatic or chemical cleavage like~ise in a manner known per se.
In this connection, the type of cleavage depends, in par-35 ticular, on the amino acid sequence of the desired pro-tein. It will be expedient in many cases to incorporate a connecting member or bridging member in the cleavage site bet~een the tendamistat sequence and the amino acid ` _ 4 _ I 33 83 3~
~
sequence of the desired protein. If the desired protein contains, for example, no methionine, the connecting mem-ber can denote Met, whereupon chemical cleavage with cyanogen chloride or bromide is carried out. If the con-necting member has a carboxyl-terminal cysteine, or if the connecting member represents Cys, it is possible for enzymatic cysteine-specific cleavage, or chemical cleav-age, for example after specific S-cyanylation, to follow.
If the bridging member has a carboxyl-terminaL tryptophan, or if the connecting member represents Trp, chemical cleavage with N-bromosuccinimide can be carried out.
Desired proteins which do not contain Asp-Pro in their a0ino acid sequence and are sufficiently stable to acid can, as fusion proteins having this bridging member, be cleaved proteolytically in a manner known per se. This results in proteins which contain N-terminal prolineand C-terminal aspartic acid. Thus, it is also possible in this way to synthesize modified proteins.
The Asp-Pro bond can be made even more labile to acid if this bridging member denotes (Asp)n-Pro or is Glu-~Asp)n-Pro, n denoting 1 to 3.
Examples of enzymatic cleavages have also been disclosed, O it also being possible to use modified enzymes having ioproved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, it is expedient to choose a peptide sequence in which an amino acid which can be eliminated by trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of the proinsulin, for example Ala-Ser-Met-Thr-Arg, since it is then possible to carry out the arginine-specific cleavage using the protease trypsin.
If the desired protein does not contain the amino acid sequence - _ 5 _ 1338338 Ile-Glu-Gly-Arg, the fusion protein having the corresponding bridging member can be cleaved with factor Xa (European Patent Application published under No. EP-A 0,025,190 on March 18, 1981 and European Patent Application published under No. EP-A 0,161,973 on November 21, 1985.
The isolation of the cleavage products depends on the properties of these proteins. Concerning the isolation of tendamistat and its derivatives, reference may be made to the literature cited in German Offenlegungsschrift 3,331,860 (published March 21, 1985, Applicant Hoechst Aktiengesellschaft).
The invention is explained in detail in the examples which follow.
Unless other~ise indicated, percentage data relate to weight. The figures having the same numbers as the examples relate thereto.
Ex~mple 1 The starting material used is the plasmid pKAI650, ~hich is described in European Patent Application 86 113 627.3 (Filed October 2, 1986, published April 15, 1987 as No. 0 218 204, Applicant Hoechst Aktiengesellschaft). This plasmid can be obtained from the plasmid pKAIl, which is described in German Offenlegungsschrift 3,331,860 (published March 21, 1985, Applicant Hoechst Aktiengesellschaft), by isolation of the 650 bp HincII/SstI fragment and cloning into the plasmid pUCl9 which has been opened with these enzymes. The unique HindIII cleavage site in this plasmid is removed (by cutting with this enzyme, filling in the protruding ends, and ligation) to result in the plasmid pKAI650a (1).
2 1~9 of (1) DNA puritied by Cstl gradient centrifugat ion are completely digested, in a 50 yl reaction mixture, uith StuI for 2 hours as stated by the manufacturer, and the en~yme is removed by phenol extraction. The lineari~ed DNA is pretipitated ~ith ethanol, redissolved and intro-duced into a ligation mixture to ~hich is added, as additional reactant, 0.1 ~9 of the chemically synthesi~ed double-stranded oligonucleotide ~2) ~hich has been . _ - 6 - 1338338 phosphorylated at the 5' end HindIII
5' C C C A A G C T T G G G 3' (2) 3' G G G T T C G A A C C C S' Transformation of the ligation mixture into E. coli JM 109 is followed by isolation of those clones ~hich harbor the recombinant plasmid pKK3a (3). The isolated plasmid DNA has a cleavage site for the restriction enzyme HindIII, ~hich permits characterization by restriction analysis. pKK3a (3) is 12 base-pairs larger than pKAI650a C~ and has a nucleotide sequence which extends the amino acid sequence by 4 amino acids, as follo~s 42 43 (44) - Glu Gly Pro Ser Leu Gly Leu 5' GAA GG C CCA AGC TTG GG C CTG 3' 3' CTT CC G GGT TCG AAC CC G GAC 5' HindIII
The other starting material used is the plasmid pYE24 (4).
This plasmid is obtained by opening the vector pUC8 ~ith EcoRI and HindIII, and ligating into this linearized plas-mid the gene for monkey proinsulin (Table 2; cf. ~etekam et al., Gene 19 (1982) 179-183).
2 ~9 of the plasmid pYE24 (4) are reacted with the re-striction enzymes EcoRI and HindIII, and the gene formonkey proinsulin is isolated by electroelution and, after purification and concentration by ethanol precipi-tation, it is ligated ~ith the synthetic DNA linker (5) 5' AGC TTG ATG GCG 3' 3' AC TAC CGC TTA A 5' (5) (HindIII) (EcoRI) The ligation product (6) is now inserted into the plasmid pKK3a (3) ~hich has been opened with HindIII, resulting in the plasmid pKK31 (7). This construction results in the following bridging member being do~nstream of the codon for amino acid Gly 43 of the tendamistat gene:
Gly Pro Ser Leu Met Ala Asn Ser Phe GGC CCA AGC TTG ATG GCG AAT TCT TTT
CCG GGT TCG AAC TAC CGC TTA AGA AAA
HindIII EcoRI
"~ 1" here, and in Table Z, designates the start of the B chain of monkey proinsulin.
In plasmid (7) the proinsulin sequence is located within - the tendamistat gene. To reconstruct this plasmid into a plasmid according to the invention, (7) is digested with SphI and SalI, and the fragment (8) is isolated. The vector pUC19 is opened ~ith SphI and SalI, and the linearized plasmid is ligated ~ith the fragment (8). The resulting plasmid pKK310 (9) codes for a fusion protein in ~hich the shortened tendamistat sequence and the linker uhich is depicted above are follo~ed only by the proinsu-lin sequence.
G The entire construction is depicted in Figure 1.
Example 2 To reconstruct the plasmid pKK310 (9) in an expression plas-mid, (9) is reacted with SstI and SphI, and the fragment (10) is isolated.
The commercially available expression vector pIJ702 (11) (obtainable from the John Innes Foundation, Nor~ich, England) is opened ~ith SphI and SstI, and the linearized plasmid (12) is ligated ~ith the fragment (10). After transformation of the strain sereptomyces lividans TK Z4 _ - 8 ~John Innes Foundation), the desired clones are identified by selection for resistance to thiostreptone. The plas-mid DNA from thiostreptone-resistant clones is isolated and examined by restriction analysis. Plasmids having the desired orientation of the gene are called pTF1 t13).
Clones which contain this recombinant plasmid secrete a protein of molecular weight 16 kD into the culture medium.
This protein shows a positive "immunoblotting" reaction with insulin antibodies (cf. Example 5).
The construction of pTF1 (13) is depicted in Figure 2.
C-v Example 3 15 The plasmid pKK3a (3), on the one hand, and the vector - pUC18, on the other hand, are each opened with HindIII, and are ligated together. The ligation mixture is used to transform the E. coli strain JM 109, which indicates successful cloning in the presence of isopropyl-B-thio-20 galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-Gal) by the formation of colorless colonies. The resultant recombinant plasmid pRS1 (14) is isolated in a manner known per se. Digestion of 1 ~9 of the plasmid with the restriction enzyme SstI, followed 25 by religation results in deletion of the pUC18 portion apart from the polylinker sequence (mcs) and the remainder of the tendamistat gene. The plasmid pRS10 (15) is ob-tained.
30 The plasmid (15) is, owing to its polylinker portion, sui~able for cloning any desired structural genes, re-sulting in plasmids which code for the corresponding fusion proteins with the shortened tendamistat sequence.
35 ~hen pRS10 (15) is digested with SphI and SstI, and the smaller fragment is isolated, the latter can be ligated into the expression vector pIJ702 in analogy to Example 2.
In this way the expression vector pTF10 (16) is obtained, 1~38338 _ _ 9 _ and this likewise, by reason of its polylinker portion, allows versatile constructions.
The construction of pTF10 (16) is depicted in Figure 3.
S
Example 4 The plasmid prE24 (4) is opened ~ith EcoRI, and the linker 5' AAT TCA AGC TTG 3' 3' GT TCG AAC TTA A 5' (EcoRI) Hind I II (EcoRI) C~
is inserted, resulting in the plasmid pYE241. Cutting with HindIII, and ligation into pKK3a (3) cut ~ith HindIII
results in the plasmid pKK32, in analogy to Example 1.
e The latter codes for a fusion protein in which the ten-damistat sequence is linked to the proinsulin sequence by the foLlo~ing bridging member:
Gly Pro Ser Leu Asn Phe Ala Arg GGC CCA AGC TTG AAT TCT GCA AGA TTT
CCG GGT TCG AAC TTA AGA CGT TCT AAA
In analogy to Example 1, pKK32 is cut ~ith SphI and SstI, and the fragment which is approximately 650 bp in size is cloned into pUC19, which has been opened ~ith these enzymes. The resulting plasmid pKK320 corresponds to plasmid pKK310 ~9) apart from the abovementioned bridging member (in which the sequence introduced by the linker is emphasized by emboldening).
In analogy to Example 2, the SstI-SphI fragment having the recombinant gene from pKK320 is cloned into pIJ702, resulting in the expression plasmid pTF2. A fusion pro-tein of 16 kD is expressed and secreted in S. lividans TK 24, and the protein reacts uith insulin antibodies (cf. Example 5). 1338338 ~ecause of the similarity of the construction of pTF2 to that of pTF1 (13), Figures 1 and 2, no depiction in a dra~ing has been given.
ExampLe 5 pKK310 (9) is partially digested with EcoRI so that only one of the two EcoRI cleavage sites is opened. After the protruding ends have been filled in using Kleno~ polymer-ase, the plasmid is religated, and the result is checked C by restriction analysis. The desired plasmid, in which ~ the EcoRI site located at the end of the proinsulin gene has been eliminated, is called pKK310a (17). Thus, the latter no~ contains a unique restrict;on site for EcoRI
in the linker region bet~een the shortened tendamistat gene and the proinsulin gene.
To construct the plasmid which codes for a fusion protein having the complete tendamistat sequence, a unique cleav-age site for KpnI is introduced, in the region of the codons for amino acids 68/69, into the DNA sequence cod-ing for tendamistat (Table 1). This entails the isolated DNA from pKAI650a (1) being digested ~ith SstI and SphI, O and the fragment which is 650 bp in size being cloned into the phage M13mp18 RF DNA, ~hich has likewise been digested ~ith these t~o enzymes, and the single-stranded DNA being prepared by knovn methods. 1 ~9 of this ssDNA
is used together ~ith 0.1 ~9 of the mutagenic "primer"
5' C GAG GTA CCG GGC GT 3' in site-directed mutagenesis (M.J. Zoller and J. Smith, Nucleic Acid Res. 10 (1982) 6487-6500).
The RF DNA is isolated from the isolated M13 clones having the mutated gene, which can be selected by the additional KpnI cleavage site, and the base exchange (C for G at the third position in the codon for Arg68) is confirmed by sequencing. Thus, the nucleotide exchange brings about no change in the amino acid sequence but does introduce S the desired new unique cleavage site into the tendamistat structural gene.
His Ala Arg Tyr Leu GTC CGG GCC ATC GAG
KpnI
C~ The mutated sequence is, after SstI-Sphl digestion, cloned 15out of the M13mp18 RF DNA into the plasmid pUC19, result-ing in the plasmid pKAI651 (18).
To check, the 650 bp SStI-SphI insert from (18) is incor-porated, as in Example 2, into the plasmid pIJ702, re-sulting in the plasmid pAX651. After this plasmid hasbeen transformed into Streptomyces lividans TK 24, the expression rates for tendamistat ~hich are obtained are the same as for the plasmid pAX650 having the unmodified tendamistat gene (German Offenlegungsschrift 3,536,182, Fig. 3).
o To prepare a plasmid, according to the invention, for a fusion protein having the entire amino acid sequence of tendamistat, the plasmid pKAI651 (18) is no~ digested ~ith SphI and KpnI, and the small fragment is ligated ~ith the linker (19) (Tyr)Leu Ala Arg Cys Leu Phe Asn Ala Met Ala Thr Gly 3' 5' CTC GCT CGC TGC CTT TTC AAT GCG ATG GCC ACC GGG
3' C ATG GAG CGA GCG ACG GAA AAG TTA CGC TAC CGG TGG CCC TTA A 5' (KpnI) (19) (EcoRI) ~ - 12 - 1338338 and the plasmid pKK310a (17) ~hich has been opened ~ith Sphl and EcoRI.
~he ligation mixture is used to transform E. coli JM 109, the plasmid DNA is isolated, and the correct fusion is verified by DNA sequencing. ~he plasmid having the correct sequence is called pKK400 (20).
~he linker (19) codes not only for the remaining amino acids of tendamistat but also for the portion of a spacer ~hich separates the tendamistat and proinsulin genes from one another, and overall embraces, ~ith the 5' end of the gene as shovn in Table 2, the codons for the follo~ing 11 amino acids:
Phe-Asn-Ala-Met-Ala-Thr-Gly-Asn-Ser-Ala-Arg Thus, the fusion protein contains in this spacer, inter alia, the amino acids methionine and arginine, ~hich per-mit cleavage vith cyanogen halide or trypsin.
The insert of about 1090 bp is isolated from the plasmidpKK400 (20) by double-digestion ~ith SstI and SphI, and the DNA is ligated into the plasmid plJ~02 (12) vhich has been opened uith the same enzymes. The result is the plasmid pGF1 (21). The ligation mixture is transformed into S. lividans TK 24, and the plasmid DNA is isolated from thiostreptone-resistant transformants ~hich have tendamistat activity. AlL positive clones contain the pGF1 Sstl-Sphl insert ~hich is 1090 bp in si~e.
The construction of pGF1 (21) is depicted in Figure 4 The tendamistat activity is determined by the plate assay which is described in Example 3 in European Patent Application 85 105 610.1 (Filed May 8, 1985, published November 21, 1985 as No. 0 161 629, Applicant Hoechst Aktiengesellschaft) and in Example 2 in European Patent Application 86 113 627.3 (Filed October 2, 1986, published April 15, 1987 as No. 0 218 204, Applicant Hoechst Aktiengesellschaft).
The fusion proteln coded for by pGFl can be expressed in a known manner. ~hen the transformed strain S. lividans TK 24 is incubated in shaken flasks at 28C for 4 days, and the mycelium is removed from the culture solution by cen-trifugation, the fusion protein can be detected in the clear solution as follows:
10 to 100 ~l of solution are mixed with 20 to 200 ~l of 15% strength trichloroacetic acid, and the precipitated protein is concentrated by centrifugation, washed and taken up in SDS-containing sample buffer (U. Laemmli, Nature 227 (1970) 680-685). After incubation at 90C for 2 minutesthe sample is separated electrophoretically on a C 10to 17% strength SDS polyacrylamide gel. A proteinof molecular weight 19 kD is obtained, that is to say in the expected molecular weight range for the fusion pro-tein composed of tendamistat and proinsulin. The fusion protein reacts both with antibodies against tendamistat and with antibodies against insulin.
Tab~e 1 - 14 - 1338338 DNA sequence (coding strand) and amino acid sequence of tendamistat 5 ' - GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG
NH - Asp Thr Thr Val Ser Glu Pro Al a Pro Ser Cys Val ACG CTC TAC CAG AGC TGG CGG TAC TCA CAG GCC GAC
Thr Leu Tyr Gln Ser Trp Arg Tyr Ser Gln Ala Asp AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC
C Asn Gly Cys Ala Glu Thr Val Thr Val Lys Val Val Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala Val GCA CCG GGC CAG ATC ACC gCC GTC GGC GAC GGC TAC
Ala Pro Gly Gln Ile Thr Thr Val Gly Asp Gly Tyr ATC GGC TCG CAC GGC CAC GCG CGC TAC CTG GCT CGC
Ile Gly Ser His Gly His Ala Arg Tyr Leu Ala Arg TGC CTT TAG-3 ' 25 Cys Leu Stp - ~ - 15 - 1338338 Tab~e 2 5 ' AAT TCT GCA AGA
3 ' GA CGT TCT
( Asn ) Ser Al a Arg (EcoRI ) B l TTT GTG AAC CAG CAC CTG TGC GGC TCC CAC CTA GTG GAA GCT CTC
AAA CAC TTG GTC GTG GAC ACG CCG AGG GTG GAT CAC CTT CGA GAG
10 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu TAC CTG GTG TGC GGG GAG CGA GGC TTC TTC TAC ACA CCC AAG ACC
ATG GAC CAC ACG CCC CTC GCT CCG AAG AAG ATG TGT GGG TTC TGG
Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr C~
2 o CGC CGG GAG GCA GAG GAC CCT CAG GTG GGG CAG GTG GAG CTG GGC
GCG GCC CTC CGT CTC CTG GGA GTC CAC CCC GTC CAC CTC GAC CCG
Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly CCC CCG GGA CCG CGT CCG TCG GAC GTC GGG AAC CGC GAC CTC CCC
Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly A l TCC CTG CAG AAG CGC GGC ATC GTG GAG CAG TGC TGC ACC AGC ATC
AGG GAC GTC TTC GCG CCG TAG CAC CTC GTC ACG ACG TGG TCG TAG
Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile .
~' TGC TCC CTC TAC CAG CTG GAG AAC TAC TGC AAC TAA TAG TCG ACC
ACG AGG GAG ATG GTC GAC CTC TTG ATG ACG TTG ATT ATC AGC TGG
40 Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn SalI
TGC AGC CA 3 ' ACG TCG GTT CGA 5 ' PstI (HindI I I ) B 1, C 1 and A 1 designate the starts of the B, C and A
chains of monkey proinsulin
5' C C C A A G C T T G G G 3' (2) 3' G G G T T C G A A C C C S' Transformation of the ligation mixture into E. coli JM 109 is followed by isolation of those clones ~hich harbor the recombinant plasmid pKK3a (3). The isolated plasmid DNA has a cleavage site for the restriction enzyme HindIII, ~hich permits characterization by restriction analysis. pKK3a (3) is 12 base-pairs larger than pKAI650a C~ and has a nucleotide sequence which extends the amino acid sequence by 4 amino acids, as follo~s 42 43 (44) - Glu Gly Pro Ser Leu Gly Leu 5' GAA GG C CCA AGC TTG GG C CTG 3' 3' CTT CC G GGT TCG AAC CC G GAC 5' HindIII
The other starting material used is the plasmid pYE24 (4).
This plasmid is obtained by opening the vector pUC8 ~ith EcoRI and HindIII, and ligating into this linearized plas-mid the gene for monkey proinsulin (Table 2; cf. ~etekam et al., Gene 19 (1982) 179-183).
2 ~9 of the plasmid pYE24 (4) are reacted with the re-striction enzymes EcoRI and HindIII, and the gene formonkey proinsulin is isolated by electroelution and, after purification and concentration by ethanol precipi-tation, it is ligated ~ith the synthetic DNA linker (5) 5' AGC TTG ATG GCG 3' 3' AC TAC CGC TTA A 5' (5) (HindIII) (EcoRI) The ligation product (6) is now inserted into the plasmid pKK3a (3) ~hich has been opened with HindIII, resulting in the plasmid pKK31 (7). This construction results in the following bridging member being do~nstream of the codon for amino acid Gly 43 of the tendamistat gene:
Gly Pro Ser Leu Met Ala Asn Ser Phe GGC CCA AGC TTG ATG GCG AAT TCT TTT
CCG GGT TCG AAC TAC CGC TTA AGA AAA
HindIII EcoRI
"~ 1" here, and in Table Z, designates the start of the B chain of monkey proinsulin.
In plasmid (7) the proinsulin sequence is located within - the tendamistat gene. To reconstruct this plasmid into a plasmid according to the invention, (7) is digested with SphI and SalI, and the fragment (8) is isolated. The vector pUC19 is opened ~ith SphI and SalI, and the linearized plasmid is ligated ~ith the fragment (8). The resulting plasmid pKK310 (9) codes for a fusion protein in ~hich the shortened tendamistat sequence and the linker uhich is depicted above are follo~ed only by the proinsu-lin sequence.
G The entire construction is depicted in Figure 1.
Example 2 To reconstruct the plasmid pKK310 (9) in an expression plas-mid, (9) is reacted with SstI and SphI, and the fragment (10) is isolated.
The commercially available expression vector pIJ702 (11) (obtainable from the John Innes Foundation, Nor~ich, England) is opened ~ith SphI and SstI, and the linearized plasmid (12) is ligated ~ith the fragment (10). After transformation of the strain sereptomyces lividans TK Z4 _ - 8 ~John Innes Foundation), the desired clones are identified by selection for resistance to thiostreptone. The plas-mid DNA from thiostreptone-resistant clones is isolated and examined by restriction analysis. Plasmids having the desired orientation of the gene are called pTF1 t13).
Clones which contain this recombinant plasmid secrete a protein of molecular weight 16 kD into the culture medium.
This protein shows a positive "immunoblotting" reaction with insulin antibodies (cf. Example 5).
The construction of pTF1 (13) is depicted in Figure 2.
C-v Example 3 15 The plasmid pKK3a (3), on the one hand, and the vector - pUC18, on the other hand, are each opened with HindIII, and are ligated together. The ligation mixture is used to transform the E. coli strain JM 109, which indicates successful cloning in the presence of isopropyl-B-thio-20 galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-Gal) by the formation of colorless colonies. The resultant recombinant plasmid pRS1 (14) is isolated in a manner known per se. Digestion of 1 ~9 of the plasmid with the restriction enzyme SstI, followed 25 by religation results in deletion of the pUC18 portion apart from the polylinker sequence (mcs) and the remainder of the tendamistat gene. The plasmid pRS10 (15) is ob-tained.
30 The plasmid (15) is, owing to its polylinker portion, sui~able for cloning any desired structural genes, re-sulting in plasmids which code for the corresponding fusion proteins with the shortened tendamistat sequence.
35 ~hen pRS10 (15) is digested with SphI and SstI, and the smaller fragment is isolated, the latter can be ligated into the expression vector pIJ702 in analogy to Example 2.
In this way the expression vector pTF10 (16) is obtained, 1~38338 _ _ 9 _ and this likewise, by reason of its polylinker portion, allows versatile constructions.
The construction of pTF10 (16) is depicted in Figure 3.
S
Example 4 The plasmid prE24 (4) is opened ~ith EcoRI, and the linker 5' AAT TCA AGC TTG 3' 3' GT TCG AAC TTA A 5' (EcoRI) Hind I II (EcoRI) C~
is inserted, resulting in the plasmid pYE241. Cutting with HindIII, and ligation into pKK3a (3) cut ~ith HindIII
results in the plasmid pKK32, in analogy to Example 1.
e The latter codes for a fusion protein in which the ten-damistat sequence is linked to the proinsulin sequence by the foLlo~ing bridging member:
Gly Pro Ser Leu Asn Phe Ala Arg GGC CCA AGC TTG AAT TCT GCA AGA TTT
CCG GGT TCG AAC TTA AGA CGT TCT AAA
In analogy to Example 1, pKK32 is cut ~ith SphI and SstI, and the fragment which is approximately 650 bp in size is cloned into pUC19, which has been opened ~ith these enzymes. The resulting plasmid pKK320 corresponds to plasmid pKK310 ~9) apart from the abovementioned bridging member (in which the sequence introduced by the linker is emphasized by emboldening).
In analogy to Example 2, the SstI-SphI fragment having the recombinant gene from pKK320 is cloned into pIJ702, resulting in the expression plasmid pTF2. A fusion pro-tein of 16 kD is expressed and secreted in S. lividans TK 24, and the protein reacts uith insulin antibodies (cf. Example 5). 1338338 ~ecause of the similarity of the construction of pTF2 to that of pTF1 (13), Figures 1 and 2, no depiction in a dra~ing has been given.
ExampLe 5 pKK310 (9) is partially digested with EcoRI so that only one of the two EcoRI cleavage sites is opened. After the protruding ends have been filled in using Kleno~ polymer-ase, the plasmid is religated, and the result is checked C by restriction analysis. The desired plasmid, in which ~ the EcoRI site located at the end of the proinsulin gene has been eliminated, is called pKK310a (17). Thus, the latter no~ contains a unique restrict;on site for EcoRI
in the linker region bet~een the shortened tendamistat gene and the proinsulin gene.
To construct the plasmid which codes for a fusion protein having the complete tendamistat sequence, a unique cleav-age site for KpnI is introduced, in the region of the codons for amino acids 68/69, into the DNA sequence cod-ing for tendamistat (Table 1). This entails the isolated DNA from pKAI650a (1) being digested ~ith SstI and SphI, O and the fragment which is 650 bp in size being cloned into the phage M13mp18 RF DNA, ~hich has likewise been digested ~ith these t~o enzymes, and the single-stranded DNA being prepared by knovn methods. 1 ~9 of this ssDNA
is used together ~ith 0.1 ~9 of the mutagenic "primer"
5' C GAG GTA CCG GGC GT 3' in site-directed mutagenesis (M.J. Zoller and J. Smith, Nucleic Acid Res. 10 (1982) 6487-6500).
The RF DNA is isolated from the isolated M13 clones having the mutated gene, which can be selected by the additional KpnI cleavage site, and the base exchange (C for G at the third position in the codon for Arg68) is confirmed by sequencing. Thus, the nucleotide exchange brings about no change in the amino acid sequence but does introduce S the desired new unique cleavage site into the tendamistat structural gene.
His Ala Arg Tyr Leu GTC CGG GCC ATC GAG
KpnI
C~ The mutated sequence is, after SstI-Sphl digestion, cloned 15out of the M13mp18 RF DNA into the plasmid pUC19, result-ing in the plasmid pKAI651 (18).
To check, the 650 bp SStI-SphI insert from (18) is incor-porated, as in Example 2, into the plasmid pIJ702, re-sulting in the plasmid pAX651. After this plasmid hasbeen transformed into Streptomyces lividans TK 24, the expression rates for tendamistat ~hich are obtained are the same as for the plasmid pAX650 having the unmodified tendamistat gene (German Offenlegungsschrift 3,536,182, Fig. 3).
o To prepare a plasmid, according to the invention, for a fusion protein having the entire amino acid sequence of tendamistat, the plasmid pKAI651 (18) is no~ digested ~ith SphI and KpnI, and the small fragment is ligated ~ith the linker (19) (Tyr)Leu Ala Arg Cys Leu Phe Asn Ala Met Ala Thr Gly 3' 5' CTC GCT CGC TGC CTT TTC AAT GCG ATG GCC ACC GGG
3' C ATG GAG CGA GCG ACG GAA AAG TTA CGC TAC CGG TGG CCC TTA A 5' (KpnI) (19) (EcoRI) ~ - 12 - 1338338 and the plasmid pKK310a (17) ~hich has been opened ~ith Sphl and EcoRI.
~he ligation mixture is used to transform E. coli JM 109, the plasmid DNA is isolated, and the correct fusion is verified by DNA sequencing. ~he plasmid having the correct sequence is called pKK400 (20).
~he linker (19) codes not only for the remaining amino acids of tendamistat but also for the portion of a spacer ~hich separates the tendamistat and proinsulin genes from one another, and overall embraces, ~ith the 5' end of the gene as shovn in Table 2, the codons for the follo~ing 11 amino acids:
Phe-Asn-Ala-Met-Ala-Thr-Gly-Asn-Ser-Ala-Arg Thus, the fusion protein contains in this spacer, inter alia, the amino acids methionine and arginine, ~hich per-mit cleavage vith cyanogen halide or trypsin.
The insert of about 1090 bp is isolated from the plasmidpKK400 (20) by double-digestion ~ith SstI and SphI, and the DNA is ligated into the plasmid plJ~02 (12) vhich has been opened uith the same enzymes. The result is the plasmid pGF1 (21). The ligation mixture is transformed into S. lividans TK 24, and the plasmid DNA is isolated from thiostreptone-resistant transformants ~hich have tendamistat activity. AlL positive clones contain the pGF1 Sstl-Sphl insert ~hich is 1090 bp in si~e.
The construction of pGF1 (21) is depicted in Figure 4 The tendamistat activity is determined by the plate assay which is described in Example 3 in European Patent Application 85 105 610.1 (Filed May 8, 1985, published November 21, 1985 as No. 0 161 629, Applicant Hoechst Aktiengesellschaft) and in Example 2 in European Patent Application 86 113 627.3 (Filed October 2, 1986, published April 15, 1987 as No. 0 218 204, Applicant Hoechst Aktiengesellschaft).
The fusion proteln coded for by pGFl can be expressed in a known manner. ~hen the transformed strain S. lividans TK 24 is incubated in shaken flasks at 28C for 4 days, and the mycelium is removed from the culture solution by cen-trifugation, the fusion protein can be detected in the clear solution as follows:
10 to 100 ~l of solution are mixed with 20 to 200 ~l of 15% strength trichloroacetic acid, and the precipitated protein is concentrated by centrifugation, washed and taken up in SDS-containing sample buffer (U. Laemmli, Nature 227 (1970) 680-685). After incubation at 90C for 2 minutesthe sample is separated electrophoretically on a C 10to 17% strength SDS polyacrylamide gel. A proteinof molecular weight 19 kD is obtained, that is to say in the expected molecular weight range for the fusion pro-tein composed of tendamistat and proinsulin. The fusion protein reacts both with antibodies against tendamistat and with antibodies against insulin.
Tab~e 1 - 14 - 1338338 DNA sequence (coding strand) and amino acid sequence of tendamistat 5 ' - GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG
NH - Asp Thr Thr Val Ser Glu Pro Al a Pro Ser Cys Val ACG CTC TAC CAG AGC TGG CGG TAC TCA CAG GCC GAC
Thr Leu Tyr Gln Ser Trp Arg Tyr Ser Gln Ala Asp AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC
C Asn Gly Cys Ala Glu Thr Val Thr Val Lys Val Val Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala Val GCA CCG GGC CAG ATC ACC gCC GTC GGC GAC GGC TAC
Ala Pro Gly Gln Ile Thr Thr Val Gly Asp Gly Tyr ATC GGC TCG CAC GGC CAC GCG CGC TAC CTG GCT CGC
Ile Gly Ser His Gly His Ala Arg Tyr Leu Ala Arg TGC CTT TAG-3 ' 25 Cys Leu Stp - ~ - 15 - 1338338 Tab~e 2 5 ' AAT TCT GCA AGA
3 ' GA CGT TCT
( Asn ) Ser Al a Arg (EcoRI ) B l TTT GTG AAC CAG CAC CTG TGC GGC TCC CAC CTA GTG GAA GCT CTC
AAA CAC TTG GTC GTG GAC ACG CCG AGG GTG GAT CAC CTT CGA GAG
10 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu TAC CTG GTG TGC GGG GAG CGA GGC TTC TTC TAC ACA CCC AAG ACC
ATG GAC CAC ACG CCC CTC GCT CCG AAG AAG ATG TGT GGG TTC TGG
Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr C~
2 o CGC CGG GAG GCA GAG GAC CCT CAG GTG GGG CAG GTG GAG CTG GGC
GCG GCC CTC CGT CTC CTG GGA GTC CAC CCC GTC CAC CTC GAC CCG
Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly CCC CCG GGA CCG CGT CCG TCG GAC GTC GGG AAC CGC GAC CTC CCC
Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly A l TCC CTG CAG AAG CGC GGC ATC GTG GAG CAG TGC TGC ACC AGC ATC
AGG GAC GTC TTC GCG CCG TAG CAC CTC GTC ACG ACG TGG TCG TAG
Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile .
~' TGC TCC CTC TAC CAG CTG GAG AAC TAC TGC AAC TAA TAG TCG ACC
ACG AGG GAG ATG GTC GAC CTC TTG ATG ACG TTG ATT ATC AGC TGG
40 Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn SalI
TGC AGC CA 3 ' ACG TCG GTT CGA 5 ' PstI (HindI I I ) B 1, C 1 and A 1 designate the starts of the B, C and A
chains of monkey proinsulin
Claims (10)
1. A process for the preparation of fusion proteins, which comprises coupling of the structural gene for the desired protein onto the 3'end of the coding strand of the tendamistat gene, which tendamistat gene may be modified, bringing about the expression of this gene structure in a Streptomycetes host cell, and isolating the secreted fusion protein from the supernatant.
2. The process as claimed in claim 1, wherein coupling is effected to the 3'end of a shortened tendamistat gene.
3. A gene structure containing the tendamistat gene, which may be modified, onto the 3'end of which a structural gene for a further protein is coupled.
4. A gene structure as claimed in claim 3, wherein the tendamistat gene is shortened at its 3'end.
5. A vector containing a gene structure as claimed in claim 3.
6. A vector containing a gene structure as claimed in claim 4.
7. A Streptomycetes cell containing a vector as claimed in claim 5.
8. A Streptomycetes cell containing a vector as claimed in claim 6.
9. A fusion protein which only has a N-terminal portion of tendamistat, which may be modified.
10. A process for the preparation of tendamistat or a modified tendamistat and a further protein, which comprises cleaving the fusion protein as claimed in claim 9, the further protein being the fusion partner in said fusion protein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873714866 DE3714866A1 (en) | 1987-05-05 | 1987-05-05 | METHOD FOR THE PRODUCTION OF FOREIGN PROTEINS IN STREPTOMYCETES |
| DEP3714866.4 | 1987-05-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1338338C true CA1338338C (en) | 1996-05-21 |
Family
ID=6326841
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000565869A Expired - Lifetime CA1338338C (en) | 1987-05-05 | 1988-05-04 | Process for the preparation of foreign proteins in streptomycetes |
Country Status (19)
| Country | Link |
|---|---|
| EP (1) | EP0289936B1 (en) |
| JP (1) | JP2671260B2 (en) |
| KR (1) | KR970001235B1 (en) |
| CN (1) | CN1029989C (en) |
| AR (1) | AR241658A1 (en) |
| AT (1) | ATE94905T1 (en) |
| AU (1) | AU612144B2 (en) |
| CA (1) | CA1338338C (en) |
| DE (2) | DE3714866A1 (en) |
| DK (1) | DK175525B1 (en) |
| ES (1) | ES2045004T3 (en) |
| FI (1) | FI97549C (en) |
| HU (1) | HU204094B (en) |
| IE (1) | IE62522B1 (en) |
| IL (1) | IL86277A0 (en) |
| NO (1) | NO178036C (en) |
| NZ (1) | NZ224464A (en) |
| PT (1) | PT87400B (en) |
| ZA (1) | ZA883168B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5426036A (en) * | 1987-05-05 | 1995-06-20 | Hoechst Aktiengesellschaft | Processes for the preparation of foreign proteins in streptomycetes |
| DE4012818A1 (en) | 1990-04-21 | 1991-10-24 | Hoechst Ag | METHOD FOR THE PRODUCTION OF FOREIGN PROTEINS IN STREPTOMYCETES |
| ATE132531T1 (en) * | 1988-11-03 | 1996-01-15 | Hoechst Ag | METHOD FOR PRODUCING AN INSULIN PREPRODUCT IN STREPTOMYCETES |
| DE3844211A1 (en) * | 1988-12-29 | 1990-07-05 | Hoechst Ag | NEW INSULINE DERIVATIVES, THE PROCESS FOR THEIR PRODUCTION, THEIR USE AND A PHARMACEUTICAL PREPARATION CONTAINING THEM |
| DE3936876A1 (en) * | 1989-11-06 | 1991-05-23 | Hoechst Ag | NEW INSULINE DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF, THEIR USE AND A PHARMACEUTICAL PREPARATION CONTAINING THE SAME |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3331860A1 (en) * | 1983-09-03 | 1985-03-21 | Hoechst Ag, 6230 Frankfurt | Process for the preparation of tendamistat |
| DE3418274A1 (en) * | 1984-05-17 | 1985-11-21 | Hoechst Ag, 6230 Frankfurt | SIGNAL PEPTIDE FOR THE EXCRETION OF PEPTIDES IN STREPTOMYCETS |
| EP0196056B1 (en) * | 1985-03-28 | 1991-05-22 | Chiron Corporation | Improved expression using fused genes providing for protein product |
| DE3707150A1 (en) * | 1987-03-06 | 1988-09-15 | Hoechst Ag | TENDAMISTAT DERIVATIVES |
| ATE132531T1 (en) * | 1988-11-03 | 1996-01-15 | Hoechst Ag | METHOD FOR PRODUCING AN INSULIN PREPRODUCT IN STREPTOMYCETES |
-
1987
- 1987-05-05 DE DE19873714866 patent/DE3714866A1/en not_active Withdrawn
-
1988
- 1988-04-28 AT AT88106786T patent/ATE94905T1/en not_active IP Right Cessation
- 1988-04-28 DE DE88106786T patent/DE3884261D1/en not_active Expired - Lifetime
- 1988-04-28 ES ES88106786T patent/ES2045004T3/en not_active Expired - Lifetime
- 1988-04-28 EP EP88106786A patent/EP0289936B1/en not_active Expired - Lifetime
- 1988-05-03 NZ NZ224464A patent/NZ224464A/en unknown
- 1988-05-03 KR KR1019880005131A patent/KR970001235B1/en not_active IP Right Cessation
- 1988-05-03 FI FI882059A patent/FI97549C/en not_active IP Right Cessation
- 1988-05-03 AR AR88310742A patent/AR241658A1/en active
- 1988-05-04 HU HU882276A patent/HU204094B/en unknown
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- 1988-05-04 PT PT87400A patent/PT87400B/en not_active IP Right Cessation
- 1988-05-04 NO NO881942A patent/NO178036C/en unknown
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- 1988-05-04 CN CN88102568A patent/CN1029989C/en not_active Expired - Lifetime
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