CA2002062A1 - Process for the preparation of an insulin precursor in streptomycetes - Google Patents
Process for the preparation of an insulin precursor in streptomycetesInfo
- Publication number
- CA2002062A1 CA2002062A1 CA002002062A CA2002062A CA2002062A1 CA 2002062 A1 CA2002062 A1 CA 2002062A1 CA 002002062 A CA002002062 A CA 002002062A CA 2002062 A CA2002062 A CA 2002062A CA 2002062 A1 CA2002062 A1 CA 2002062A1
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- Prior art keywords
- gene
- lys
- tendamistat
- insulin
- fusion protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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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
-
- 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
-
- 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)
-
- 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
-
- 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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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Diabetes (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Endocrinology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Abstract of the disclosure HOE 88/F 313 K
A process fox the preparation of an insulin precursor in Streptomycetes When a gene for a shortened proinsulin in which the insulin B chain is linked to the A chain only via Lys or Lys-Lys is coupled to the tendamistat gene, this gene construction is introduced into an expression vector, and the latter is used to transform a Streptomycetes host cell, there is expression and secretion of the corres-ponding fusion protein. The fusion protein can easily be cleaved to give insulin precursors.
A process fox the preparation of an insulin precursor in Streptomycetes When a gene for a shortened proinsulin in which the insulin B chain is linked to the A chain only via Lys or Lys-Lys is coupled to the tendamistat gene, this gene construction is introduced into an expression vector, and the latter is used to transform a Streptomycetes host cell, there is expression and secretion of the corres-ponding fusion protein. The fusion protein can easily be cleaved to give insulin precursors.
Description
Z~ Z
HOECHST AKTIENGESELLSCHAET HOE 88/F 313 K Dr.KL/PP
Description A process for the preparation of an insulin precursor in Streptomycetes A process for the preparation of fusion proteins has already been proposed ~hich comprises C-terminal coupling of the structural gene for the desired protein to the optionally modified tendamistat gene, brinying about the expression of this gene structure in a Streptomycetes host cell, and isolating the secreted fusion protein from the culture supernatant (DE 37 14 866 A1 or EP 0,289,936 A2). The older application additionally relates to gene structures containing the optionally modified tendamistat gene to which a structural gene for another protein is C-terminally coupled, vectors which contain a gene ~truc-ture of this type, Streptomycetes cells which contain a vector of this type, and fusion proteins which have an N-terminal portion of optionally modified tendamistat. The older application contains examples of fusion proteins in which the amino acid sequence of tendamistat is coupled via a bridging member to that of monkey proinsulin.
In a further development of the concept of this invention it has now been found that this process can be u~ed particularly well to prepare a fu~ion protein in which the tendamistat portion is followed by a shortened proinsulin whose C chain comprises only one or two ly~ine residues. These precursors can be converted particularly straightforwardly and economically into human insulin.
Particular embodiments of the invention relate to advan-tageous gene tructures for the amplification and expres-sion of the gene which codes for the fusion ~rotein.
Further preferred embodiments of the invention are explained hereinafter and defined in the patent claims.
HOECHST AKTIENGESELLSCHAET HOE 88/F 313 K Dr.KL/PP
Description A process for the preparation of an insulin precursor in Streptomycetes A process for the preparation of fusion proteins has already been proposed ~hich comprises C-terminal coupling of the structural gene for the desired protein to the optionally modified tendamistat gene, brinying about the expression of this gene structure in a Streptomycetes host cell, and isolating the secreted fusion protein from the culture supernatant (DE 37 14 866 A1 or EP 0,289,936 A2). The older application additionally relates to gene structures containing the optionally modified tendamistat gene to which a structural gene for another protein is C-terminally coupled, vectors which contain a gene ~truc-ture of this type, Streptomycetes cells which contain a vector of this type, and fusion proteins which have an N-terminal portion of optionally modified tendamistat. The older application contains examples of fusion proteins in which the amino acid sequence of tendamistat is coupled via a bridging member to that of monkey proinsulin.
In a further development of the concept of this invention it has now been found that this process can be u~ed particularly well to prepare a fu~ion protein in which the tendamistat portion is followed by a shortened proinsulin whose C chain comprises only one or two ly~ine residues. These precursors can be converted particularly straightforwardly and economically into human insulin.
Particular embodiments of the invention relate to advan-tageous gene tructures for the amplification and expres-sion of the gene which codes for the fusion ~rotein.
Further preferred embodiments of the invention are explained hereinafter and defined in the patent claims.
2~ 6Z
The gene structure employed according to the invention is advantageously based on a synthetic gene which codes for the shortened proinsulin derivative. It iB expedient in the construction of this gene to take account of the S specific codon usage of Streptomycetes. It has emerged that the yield of fusion protein is thereby increased.
It is also advantageous to incorporate a terminator ~equence in the synthetic gene structure, because an increase in the synthesis rate is also thereby achieved.
A great advantage of the process according to the inven-tion comprises the possibility of detecting the fusion proteins using the plate test which is described in EP-Al 0,161,629 in Example 3 and in German Offenlegungs-schrift 3,536,182. This considerably facilitates not only the selection of the interesting clones but also the working up because the effect of the different parameters on the yield can easily be established.
The fusion proteins obtained according to the invention are apparently present in a conformation which corres-ponds, or at least approximates, to that of matureinsulin. This not only considerably facilitates the further processing to insulin but, moreover, at fermenta-tion times long enough to provide a good yield, surpris-ingly there is no noticeable attack by the proteases excreted into the fermentation medium.
The modification according to the invention of the proinsulin molecule with its ~hortened C chain permits straightforward proces~ing to human insulin, namely by chemical cleavage with hydroxylamine and/or by enzymatic cleavage using trypsin or, advantageously, lysyl endo-proteinases. Enzymatic cleavage is preferred. Lysyl endoproteinases carry out specific carboxyl-terminal cleavage after the amino acid lysine. The favorable arrangement of the A and the B chain in the fusion protein according to the invention means that the action :-..
. ::.
, ~
2~ CP~
of the said enzymes results in an insulin pr~cursor inwhich, surprisingly, the disulfide bridge~ are correctly linked.
It is expedient in the construction of the gene to provide between the tendamistat portion and the start of the proinsulin molecule a bridging member which permits the proinsulin derivative to be cleaved off from the tendamistat portion with the same enzyme used to cleave the proinsulin derivative into the two insulin chainsO
Cleavage of ~he fusion proteins according to the inven-tion with a lysyl endopeptidase result~ - depending on the construction of the modified C chain - in de-B30-insulin which can be transformed into human insulin by transpeptidation, or B31-Lys-insulin or B3l-Lys-B32-Lys-insulin, each of which can be transformed into humaninsulin, for example, by the use of carboxypeptidase B.
Particularly high yields of the de~ired protein are obtained when gene constructions with a shortened ten-damistat gene are employed. This embodiment of the proce~s according to the invention has the great advan-tage that the portion o the modified insulin in the fusion protein comprises about one half, and thus con-tains considerably less "ballast". The correct folding of the fusion pxotein i8 not impaired by the shortaning of the tendamistat portion, BO that the advantageous working Up i8 therefore also possible with the smaller fusion protein according to the invention. Nor is this advantage achieved at the expense of an increased rate of degra-dation by the proteases intrinsic to the host - in fact, unexpectedly, it has emerged that the stability to these protesses i~ increased.
The invention thu~ allows a whole series of advantageous gene constructions which result in insulin precursors which can easily be separated from the "balla~t portion"
of the fusion protein. This straiqhtforward working up Z~2('62 additionally improves the yield of human insulin.
The separation of the fusion protein from the culture medium, it6 further processing to the insulin precursor and the transformation thereof into human insulin can be carried out by methods known per se. Thus, the fusion protein can advantageously be isolated by adsorption or ion exchange chromatography and/or gel filtration, and the proinsulin portion can be cleaved off chemically or, advantageously, enzymatically. The construction of appropriate bridging members is generally known and described, for example, in EP-A2 0,229,998.
EP-B 0,089,007 discloses analogs of prepro- and pro-insulin which carry at the C end of the prechain (or at the N-terminus of proinsulin) Lys or Arg (which is also preferred in the constructions according to the inven-tion), whose B chain terminates with B2~-Lys and where the C peptide can be shortened to Lys or Arg 80 that there-fore B2~-Lys in the proinsulin structure is, in the simple6t case, followed by only Lys or Arg, to which the A chain is attached. These compounds are used a6 precur-~ors for preparing insulins with the aid of trypsin or trypsin-like endopeptidases and of an ester of a natural amino acid which, where appropriate, carries protective groups.
Insulin precursors in which the B and A chain are con-nected by the bridging member -X-Y-, in which X snd Y are identical or different and represent Lys and Arg, are disclosed in EP-A 0,195,691. These insulin precursors are expressed from yeast and then converted into human insulin by enzymatic transformation. Insulin precursors with a shortened C chain are al~o disclosed in EP-A
0,163,529. EP-B 0,132,769 and 0,132,770 describe insulin derivatives and pharmaceutical agents containing them.
.
The invention is illustrated in more detail in the examples which follow. Unless stated otherwise, ' ' ~ :' ,. ; ' ~
percentage data relate to weight.
The figure illustrateæ the gene construction according to the invention in Example 1. It is not true to ~cale.
Example 1 The synthetic gene (1) depicted in ~able 1 is chemically synthesized in a manner known per se by the phosphoami-dite method. In the codon selection account was taken of the preference of Streptomycetes for G and C. As with the gene coding for monkey proinsulin ~n the earlier application (Table 2 therein), the gene (1) ~hown in Table 1 also has at the S' end a protruding sequence typical for the restriction enzyme EcoRI. The structural gene is followed by two stop codons and a linker sequence with the recog-nition site for the enzyme SalI. The protruding sequence corresponding to the restriction enzyme HindIII is located at the 3' end.
The commercially available plasmid pUC19 is cut with the enzymes ~coRI and HindIII, and the synthetic gene (1) shown in Ta~le 1 i8 ligated in. The result is the plasmid pll (2). After amplification, the synthetic gene is cut out a~ fragment (3) with the enzyme~ EcoRI and SalI and employed for t:he construction describ~d hereinafter.
The plasmid pUC19 i8 completely digested with SmaI and ligated with the terminator sequence (4) depicted in Tabla 2. Pla~mids which contain this sequence in the correct orientation are called pTl (5). This plasmid (5) is opened with EcoRI, and the cleavage site is filled in with DNA polymerase (Klenow fragment). The plasmid pT2 (6) is obtained by religation. This plasmid i~ opened with the enzymes SalI and SphI, and the large fragment ( 7 ) i8 isolated.
The plasmid pK~400 (8) Icf. earlier application, Figure 4, (20)) i~ cut with SphI and EcoRIt and the small fragment (9~ with the tendamistat gene is isolated.
Ligation of fragments (3), (7~ and (93 re~ult6 in the plasmid pRK500 (10) in which the tendamistat ~equence is followed by the bridging member Phe Asn Al a Met Al a Thr Gl y Asn Ser Asn Gl y Lys TTC AAT GCG ATG GCC ACC GGG ATT TC~ AAC GGC AAG
AAG TTA CGC TAC CGG TGG CCC TAA AGC TTG 1: CG TTC
EcoR I
coding for 12 amino acids, and then by the gene for the proinsulin modified according to the invention. The correct arrangement is checked by cutting with SphI and SstI, resulting in a fragment of 833 bp from the plasmid about 3.5 kb in size. The sequence i8 confirmed as correct by DNA sequencing using the dideoxy method~
Gene constructions, according to the invention, in which the Lys acting a8 C peptide is supplemented by another Lys are prepared analogously. For thi~ purpose, the triplet AAG coding for Lys is doubled. The plasmid pI2, and therefrom the vector pKR600, are obtained analo-gously.
Example 2 In analogy to the vector pGF1 provosed m ~e earlier application, the expression plasmids pGF2 and pGF3 are prepared from the vectors pKX500 and pKR600. For this purpose, double digestion with SphI and SstI of each of the vec~ors pRK500 and pRK600 is carried out to isolate the insert of 823 and 826 bp respectively, and these DNA
fra~ments are ligated into the exprQssion plasmid pIJ702 cleaved with the same enzymes. The ligation mi~ture is transfonmed into S. lividan~ TK 24, and the plasmid DNA
is isolated from thiostrepton resistant transformants which show tendamistat activity (plate test). All the positive clones contain the insert from p~500 or pRK600 employed.
~ 7 --The expres~ion of the coded fu~ion protein can be carried out in a known manner. If the transformed strain S.
lividans TK 24 is incubated in a shaken flask at 28~C for four days and the mycelium is separated from the culture solution by centrifugation~ the fusion protein can be detected in the clear solution as follows:
20 to 200 ~1 of 15~ ~trength trichloroacetic acid are added to 10 to 100 ~1 of solution, and the precipitated protein is concentrated by centrifugation, washed and taken up in SDS-containing sample buffer (U. Laemmli, Nature 227 (1970~ 680-685). Incubation at 90~C for 2 minutes is followed by fractionation by electrophoresis on a 10-17~ SDS polyacrylamide gel. A protein of molecu-lar weight 15 kD is obtained, that is to say in the molecular weight range expected for the fusion protein composed of tendamistat and proin~ulin. The fu ion protein reacts both with antibodies against tend~mistat and wi~h antibodies against insulin.
~xample 3 The expres~ion vector pTF2 (DE 37 14 866 A1, E~ample 4) i8 digested with the restriction enzymes EcoRI and SstI, snd the fra~ent which encodes monkey proinsulin is removed. The fragment 5.65 kbp in size i8 used for the ligation reac1:ion described below.
These ~ame restriction enzymes are used to cut a DNA
fragment which i6 285 bp in size and which contains the shortened proinsulin gene, as well as the termination sequence, out of the plasmid pRK500 (Example 1~.
Ligation of the fragment 5.65 kbp in size from pTF2 with the fragment 285 bp in size from pKK500 yields the expres~ion plasmid pTF3.
Transformation of protoplasts of Streptomyces lividans TK24 with the ligation mixture results in clones which are thiostrepton-resistant and secrete 8 fusion protein which reacts with antibodies against proinsulin. This fusion protein comprises the first 41 amino acids of tendamistat, the bridging member Pro-Ser-Leu-Asn-Ser-Asn-Gly-Lys and the shortened proinsulin.
2~ 2 Table 1 Bl 10 ASN SER ASN GLY LYS PHE VAL ASN GLN HIS LEU CYS GLY SER HIS
AAT TCG AAC GGC AAG TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC
GC TTG CCG TTC AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG
(EcoRI) LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARG GLY PHE PHE
CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC
GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG
C Al 40 TAC ACC CCC AAG ACC AAG GGC ATC GTG GAG CAG TGC TGT ACG TCC
ILE CYS SER LEU TYR GLN LEU GLU ASN TYR CYS ASN STP STP
ATC TGC TCC CTC TAC CAG CTC GAG AAC TAC TGC AAC TAG TAA
TAG ACG AGG GAG ATG GTC GAG CTC TTG ATG ACG TTG ATC ATT
CTC GAC CTG CAG CCA
CAG CTG GAC GTC GGT TCG A
SalI (HlndIII) Table 2 : 5'-CGATAAACCGATACAATTAAAGGCTCCr~GAGCClllTrrl~GACATTTTCAACGTGGATC
GCTATTTGGCTATGTTAATTTCCGAGGAAAACCTCGGAAAAAAAAACCTCTAAAAGTTGCACCTAG-5' . ' ' . - . ~
. , - . .
.
,
The gene structure employed according to the invention is advantageously based on a synthetic gene which codes for the shortened proinsulin derivative. It iB expedient in the construction of this gene to take account of the S specific codon usage of Streptomycetes. It has emerged that the yield of fusion protein is thereby increased.
It is also advantageous to incorporate a terminator ~equence in the synthetic gene structure, because an increase in the synthesis rate is also thereby achieved.
A great advantage of the process according to the inven-tion comprises the possibility of detecting the fusion proteins using the plate test which is described in EP-Al 0,161,629 in Example 3 and in German Offenlegungs-schrift 3,536,182. This considerably facilitates not only the selection of the interesting clones but also the working up because the effect of the different parameters on the yield can easily be established.
The fusion proteins obtained according to the invention are apparently present in a conformation which corres-ponds, or at least approximates, to that of matureinsulin. This not only considerably facilitates the further processing to insulin but, moreover, at fermenta-tion times long enough to provide a good yield, surpris-ingly there is no noticeable attack by the proteases excreted into the fermentation medium.
The modification according to the invention of the proinsulin molecule with its ~hortened C chain permits straightforward proces~ing to human insulin, namely by chemical cleavage with hydroxylamine and/or by enzymatic cleavage using trypsin or, advantageously, lysyl endo-proteinases. Enzymatic cleavage is preferred. Lysyl endoproteinases carry out specific carboxyl-terminal cleavage after the amino acid lysine. The favorable arrangement of the A and the B chain in the fusion protein according to the invention means that the action :-..
. ::.
, ~
2~ CP~
of the said enzymes results in an insulin pr~cursor inwhich, surprisingly, the disulfide bridge~ are correctly linked.
It is expedient in the construction of the gene to provide between the tendamistat portion and the start of the proinsulin molecule a bridging member which permits the proinsulin derivative to be cleaved off from the tendamistat portion with the same enzyme used to cleave the proinsulin derivative into the two insulin chainsO
Cleavage of ~he fusion proteins according to the inven-tion with a lysyl endopeptidase result~ - depending on the construction of the modified C chain - in de-B30-insulin which can be transformed into human insulin by transpeptidation, or B31-Lys-insulin or B3l-Lys-B32-Lys-insulin, each of which can be transformed into humaninsulin, for example, by the use of carboxypeptidase B.
Particularly high yields of the de~ired protein are obtained when gene constructions with a shortened ten-damistat gene are employed. This embodiment of the proce~s according to the invention has the great advan-tage that the portion o the modified insulin in the fusion protein comprises about one half, and thus con-tains considerably less "ballast". The correct folding of the fusion pxotein i8 not impaired by the shortaning of the tendamistat portion, BO that the advantageous working Up i8 therefore also possible with the smaller fusion protein according to the invention. Nor is this advantage achieved at the expense of an increased rate of degra-dation by the proteases intrinsic to the host - in fact, unexpectedly, it has emerged that the stability to these protesses i~ increased.
The invention thu~ allows a whole series of advantageous gene constructions which result in insulin precursors which can easily be separated from the "balla~t portion"
of the fusion protein. This straiqhtforward working up Z~2('62 additionally improves the yield of human insulin.
The separation of the fusion protein from the culture medium, it6 further processing to the insulin precursor and the transformation thereof into human insulin can be carried out by methods known per se. Thus, the fusion protein can advantageously be isolated by adsorption or ion exchange chromatography and/or gel filtration, and the proinsulin portion can be cleaved off chemically or, advantageously, enzymatically. The construction of appropriate bridging members is generally known and described, for example, in EP-A2 0,229,998.
EP-B 0,089,007 discloses analogs of prepro- and pro-insulin which carry at the C end of the prechain (or at the N-terminus of proinsulin) Lys or Arg (which is also preferred in the constructions according to the inven-tion), whose B chain terminates with B2~-Lys and where the C peptide can be shortened to Lys or Arg 80 that there-fore B2~-Lys in the proinsulin structure is, in the simple6t case, followed by only Lys or Arg, to which the A chain is attached. These compounds are used a6 precur-~ors for preparing insulins with the aid of trypsin or trypsin-like endopeptidases and of an ester of a natural amino acid which, where appropriate, carries protective groups.
Insulin precursors in which the B and A chain are con-nected by the bridging member -X-Y-, in which X snd Y are identical or different and represent Lys and Arg, are disclosed in EP-A 0,195,691. These insulin precursors are expressed from yeast and then converted into human insulin by enzymatic transformation. Insulin precursors with a shortened C chain are al~o disclosed in EP-A
0,163,529. EP-B 0,132,769 and 0,132,770 describe insulin derivatives and pharmaceutical agents containing them.
.
The invention is illustrated in more detail in the examples which follow. Unless stated otherwise, ' ' ~ :' ,. ; ' ~
percentage data relate to weight.
The figure illustrateæ the gene construction according to the invention in Example 1. It is not true to ~cale.
Example 1 The synthetic gene (1) depicted in ~able 1 is chemically synthesized in a manner known per se by the phosphoami-dite method. In the codon selection account was taken of the preference of Streptomycetes for G and C. As with the gene coding for monkey proinsulin ~n the earlier application (Table 2 therein), the gene (1) ~hown in Table 1 also has at the S' end a protruding sequence typical for the restriction enzyme EcoRI. The structural gene is followed by two stop codons and a linker sequence with the recog-nition site for the enzyme SalI. The protruding sequence corresponding to the restriction enzyme HindIII is located at the 3' end.
The commercially available plasmid pUC19 is cut with the enzymes ~coRI and HindIII, and the synthetic gene (1) shown in Ta~le 1 i8 ligated in. The result is the plasmid pll (2). After amplification, the synthetic gene is cut out a~ fragment (3) with the enzyme~ EcoRI and SalI and employed for t:he construction describ~d hereinafter.
The plasmid pUC19 i8 completely digested with SmaI and ligated with the terminator sequence (4) depicted in Tabla 2. Pla~mids which contain this sequence in the correct orientation are called pTl (5). This plasmid (5) is opened with EcoRI, and the cleavage site is filled in with DNA polymerase (Klenow fragment). The plasmid pT2 (6) is obtained by religation. This plasmid i~ opened with the enzymes SalI and SphI, and the large fragment ( 7 ) i8 isolated.
The plasmid pK~400 (8) Icf. earlier application, Figure 4, (20)) i~ cut with SphI and EcoRIt and the small fragment (9~ with the tendamistat gene is isolated.
Ligation of fragments (3), (7~ and (93 re~ult6 in the plasmid pRK500 (10) in which the tendamistat ~equence is followed by the bridging member Phe Asn Al a Met Al a Thr Gl y Asn Ser Asn Gl y Lys TTC AAT GCG ATG GCC ACC GGG ATT TC~ AAC GGC AAG
AAG TTA CGC TAC CGG TGG CCC TAA AGC TTG 1: CG TTC
EcoR I
coding for 12 amino acids, and then by the gene for the proinsulin modified according to the invention. The correct arrangement is checked by cutting with SphI and SstI, resulting in a fragment of 833 bp from the plasmid about 3.5 kb in size. The sequence i8 confirmed as correct by DNA sequencing using the dideoxy method~
Gene constructions, according to the invention, in which the Lys acting a8 C peptide is supplemented by another Lys are prepared analogously. For thi~ purpose, the triplet AAG coding for Lys is doubled. The plasmid pI2, and therefrom the vector pKR600, are obtained analo-gously.
Example 2 In analogy to the vector pGF1 provosed m ~e earlier application, the expression plasmids pGF2 and pGF3 are prepared from the vectors pKX500 and pKR600. For this purpose, double digestion with SphI and SstI of each of the vec~ors pRK500 and pRK600 is carried out to isolate the insert of 823 and 826 bp respectively, and these DNA
fra~ments are ligated into the exprQssion plasmid pIJ702 cleaved with the same enzymes. The ligation mi~ture is transfonmed into S. lividan~ TK 24, and the plasmid DNA
is isolated from thiostrepton resistant transformants which show tendamistat activity (plate test). All the positive clones contain the insert from p~500 or pRK600 employed.
~ 7 --The expres~ion of the coded fu~ion protein can be carried out in a known manner. If the transformed strain S.
lividans TK 24 is incubated in a shaken flask at 28~C for four days and the mycelium is separated from the culture solution by centrifugation~ the fusion protein can be detected in the clear solution as follows:
20 to 200 ~1 of 15~ ~trength trichloroacetic acid are added to 10 to 100 ~1 of solution, and the precipitated protein is concentrated by centrifugation, washed and taken up in SDS-containing sample buffer (U. Laemmli, Nature 227 (1970~ 680-685). Incubation at 90~C for 2 minutes is followed by fractionation by electrophoresis on a 10-17~ SDS polyacrylamide gel. A protein of molecu-lar weight 15 kD is obtained, that is to say in the molecular weight range expected for the fusion protein composed of tendamistat and proin~ulin. The fu ion protein reacts both with antibodies against tend~mistat and wi~h antibodies against insulin.
~xample 3 The expres~ion vector pTF2 (DE 37 14 866 A1, E~ample 4) i8 digested with the restriction enzymes EcoRI and SstI, snd the fra~ent which encodes monkey proinsulin is removed. The fragment 5.65 kbp in size i8 used for the ligation reac1:ion described below.
These ~ame restriction enzymes are used to cut a DNA
fragment which i6 285 bp in size and which contains the shortened proinsulin gene, as well as the termination sequence, out of the plasmid pRK500 (Example 1~.
Ligation of the fragment 5.65 kbp in size from pTF2 with the fragment 285 bp in size from pKK500 yields the expres~ion plasmid pTF3.
Transformation of protoplasts of Streptomyces lividans TK24 with the ligation mixture results in clones which are thiostrepton-resistant and secrete 8 fusion protein which reacts with antibodies against proinsulin. This fusion protein comprises the first 41 amino acids of tendamistat, the bridging member Pro-Ser-Leu-Asn-Ser-Asn-Gly-Lys and the shortened proinsulin.
2~ 2 Table 1 Bl 10 ASN SER ASN GLY LYS PHE VAL ASN GLN HIS LEU CYS GLY SER HIS
AAT TCG AAC GGC AAG TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC
GC TTG CCG TTC AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG
(EcoRI) LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARG GLY PHE PHE
CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC
GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG
C Al 40 TAC ACC CCC AAG ACC AAG GGC ATC GTG GAG CAG TGC TGT ACG TCC
ILE CYS SER LEU TYR GLN LEU GLU ASN TYR CYS ASN STP STP
ATC TGC TCC CTC TAC CAG CTC GAG AAC TAC TGC AAC TAG TAA
TAG ACG AGG GAG ATG GTC GAG CTC TTG ATG ACG TTG ATC ATT
CTC GAC CTG CAG CCA
CAG CTG GAC GTC GGT TCG A
SalI (HlndIII) Table 2 : 5'-CGATAAACCGATACAATTAAAGGCTCCr~GAGCClllTrrl~GACATTTTCAACGTGGATC
GCTATTTGGCTATGTTAATTTCCGAGGAAAACCTCGGAAAAAAAAACCTCTAAAAGTTGCACCTAG-5' . ' ' . - . ~
. , - . .
.
,
Claims (8)
1. A process for the preparation of a fusion protein which comprises coupling the structural gene coding for a proinsulin derivative in which the B chain is connected to the A chain via a bridging member coding for Lys or Lys-Lys to the C terminus of the tendamistat gene, expressing this gene structure in a Streptomycetes host cell and isolating the secreted fusion protein from the supernatant.
2. A process as claimed in claim 1, wherein the tendamistat gene is modified.
3. A process as claimed in claim 1 or 2, wherein the tendamistat gene is shortened.
4. A gene structure containing the optionally modified tendamistat gene to which the structural gene defined in claim 1 is coupled C-terminally.
5. A vector containing a gene structure as claimed in claim 4.
6. A Streptomycetes cell containing a vector as claimed in claim 5.
7. A fusion protein which has a N-terminal portion of optionally modified tendamistat to whose C terminus the proinsulin defined in cliam 1 is bonded.
8. The process as claimed in claim 1, and substantially as described herein.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3837273A DE3837273A1 (en) | 1987-05-05 | 1988-11-03 | Process for the production of an insulin precursor in streptomyces |
| DEP3837273.8 | 1988-11-03 | ||
| DEP3927449.7 | 1989-08-19 | ||
| DE19893927449 DE3927449A1 (en) | 1989-08-19 | 1989-08-19 | New insulin fusion proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2002062A1 true CA2002062A1 (en) | 1990-05-03 |
Family
ID=25873834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002002062A Abandoned CA2002062A1 (en) | 1988-11-03 | 1989-11-02 | Process for the preparation of an insulin precursor in streptomycetes |
Country Status (18)
| Country | Link |
|---|---|
| EP (1) | EP0367163B1 (en) |
| JP (1) | JPH02219588A (en) |
| KR (1) | KR900008040A (en) |
| CN (1) | CN1042378A (en) |
| AT (1) | ATE132531T1 (en) |
| AU (1) | AU619054B2 (en) |
| CA (1) | CA2002062A1 (en) |
| DE (1) | DE58909556D1 (en) |
| DK (1) | DK546889A (en) |
| ES (1) | ES2081826T3 (en) |
| FI (1) | FI95600C (en) |
| GR (1) | GR3018738T3 (en) |
| HU (1) | HU209596B (en) |
| IE (1) | IE893530L (en) |
| IL (1) | IL92178A (en) |
| NO (1) | NO894363L (en) |
| NZ (1) | NZ231222A (en) |
| PT (1) | PT92177B (en) |
Families Citing this family (6)
| 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 |
| DE3714866A1 (en) * | 1987-05-05 | 1988-11-24 | Hoechst Ag | METHOD FOR THE PRODUCTION OF FOREIGN PROTEINS IN STREPTOMYCETES |
| FI103805B (en) | 1990-09-05 | 1999-09-30 | Hoechst Ag | Process for hydrolysis of preproinsulins |
| GB9513967D0 (en) * | 1995-07-08 | 1995-09-06 | Univ Leicester | Insulin |
| BRPI0823004B8 (en) | 2008-08-07 | 2021-05-25 | Biocon Ltd | process for preparing insulin compounds |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3418274A1 (en) * | 1984-05-17 | 1985-11-21 | Hoechst Ag, 6230 Frankfurt | SIGNAL PEPTIDE FOR THE EXCRETION OF PEPTIDES IN STREPTOMYCETS |
| DK129385A (en) * | 1985-03-22 | 1986-09-23 | Novo Industri As | PEPTIDES AND PREPARATION THEREOF |
| DK437786D0 (en) * | 1986-09-12 | 1986-09-12 | Nordisk Gentofte | insulin precursors |
| DE3707150A1 (en) * | 1987-03-06 | 1988-09-15 | Hoechst Ag | TENDAMISTAT DERIVATIVES |
| DE3714866A1 (en) * | 1987-05-05 | 1988-11-24 | Hoechst Ag | METHOD FOR THE PRODUCTION OF FOREIGN PROTEINS IN STREPTOMYCETES |
-
1989
- 1989-10-28 ES ES89120056T patent/ES2081826T3/en not_active Expired - Lifetime
- 1989-10-28 EP EP89120056A patent/EP0367163B1/en not_active Expired - Lifetime
- 1989-10-28 DE DE58909556T patent/DE58909556D1/en not_active Expired - Fee Related
- 1989-10-28 AT AT89120056T patent/ATE132531T1/en not_active IP Right Cessation
- 1989-11-01 IL IL9217889A patent/IL92178A/en not_active IP Right Cessation
- 1989-11-01 FI FI895184A patent/FI95600C/en not_active IP Right Cessation
- 1989-11-01 AU AU43950/89A patent/AU619054B2/en not_active Ceased
- 1989-11-01 NZ NZ231222A patent/NZ231222A/en unknown
- 1989-11-02 HU HU895649A patent/HU209596B/en not_active IP Right Cessation
- 1989-11-02 CA CA002002062A patent/CA2002062A1/en not_active Abandoned
- 1989-11-02 NO NO89894363A patent/NO894363L/en unknown
- 1989-11-02 JP JP1287282A patent/JPH02219588A/en active Pending
- 1989-11-02 KR KR1019890015844A patent/KR900008040A/en active IP Right Grant
- 1989-11-02 DK DK546889A patent/DK546889A/en not_active Application Discontinuation
- 1989-11-02 PT PT92177A patent/PT92177B/en not_active IP Right Cessation
- 1989-11-02 IE IE893530A patent/IE893530L/en unknown
- 1989-11-03 CN CN89108320A patent/CN1042378A/en active Pending
-
1996
- 1996-01-19 GR GR960400123T patent/GR3018738T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FI895184A0 (en) | 1989-11-01 |
| JPH02219588A (en) | 1990-09-03 |
| HU895649D0 (en) | 1990-01-28 |
| IL92178A (en) | 1995-11-27 |
| KR900008040A (en) | 1990-06-02 |
| GR3018738T3 (en) | 1996-04-30 |
| AU4395089A (en) | 1990-05-10 |
| FI95600C (en) | 1996-02-26 |
| EP0367163B1 (en) | 1996-01-03 |
| EP0367163A3 (en) | 1991-03-06 |
| PT92177A (en) | 1990-05-31 |
| AU619054B2 (en) | 1992-01-16 |
| DK546889D0 (en) | 1989-11-02 |
| DK546889A (en) | 1990-05-04 |
| IL92178A0 (en) | 1990-07-26 |
| DE58909556D1 (en) | 1996-02-15 |
| HUT53675A (en) | 1990-11-28 |
| ATE132531T1 (en) | 1996-01-15 |
| EP0367163A2 (en) | 1990-05-09 |
| ES2081826T3 (en) | 1996-03-16 |
| IE893530L (en) | 1990-05-03 |
| NO894363L (en) | 1990-05-04 |
| HU209596B (en) | 1994-08-29 |
| NO894363D0 (en) | 1989-11-02 |
| PT92177B (en) | 1995-06-30 |
| CN1042378A (en) | 1990-05-23 |
| FI95600B (en) | 1995-11-15 |
| NZ231222A (en) | 1991-08-27 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |