IE59127B1 - Fusion proteins, a process for their preparation oand their use - Google Patents
Fusion proteins, a process for their preparation oand their useInfo
- Publication number
- IE59127B1 IE59127B1 IE197786A IE197786A IE59127B1 IE 59127 B1 IE59127 B1 IE 59127B1 IE 197786 A IE197786 A IE 197786A IE 197786 A IE197786 A IE 197786A IE 59127 B1 IE59127 B1 IE 59127B1
- Authority
- IE
- Ireland
- Prior art keywords
- sequence
- fusion protein
- amino acids
- plasmid
- protein
- Prior art date
Links
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- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
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- 125000001302 tertiary amino group Chemical group 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
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- 238000013519 translation Methods 0.000 description 1
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Classifications
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Abstract
1. Claims for the Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE A fusion protein of the general formula Met - Xn - D' - Y - Z in which n is zero or 1, X is a sequence of 1 to 12 genetically codable amino acids, D' is a sequence of about 70 amino acids in the region of the sequence of amino acids 23 - 93 of the D-peptide in the trp operon of E. coli, Y denotes a sequence of one or more genetically codable amino acids which permits the following amino acid sequence Z to be cleaved off, and Z is a sequence of genetically codable amino acids. 1. Claims for the Contracting State : AT A process for the preparation of a fusion protein of the general formula (1) Met - Xn - D' - Y - Z in which n is zero or 1, X is a sequence of 1 to 12 genetically codable amino acids, D' is a sequence of about 70 amino acids in the region of the sequence of amino acids 23 - 93 of the D-peptide in the trp operon of E. coli, Y denotes a sequence of one or more genetically codable amino acids which permits the following amino acid sequence Z to be cleaved off, and Z is a sequence of genetically codable amino acids, characterized by expressing a gene structure which codes for these fusion proteins in a host cell, and separating the fusion protein.
Description
tively small eukaryotic proteins with a aoleculpr weight * up t© about *35000 baltons tEie yield obtained in bacteria is frequently only small- St is presumed that the proteins 5 which are formed are rapidly degraded by preteases intrinsic to the host» For this reason, proteins of this type ar© advantageously prepared as fusion proteins, in particular having a portion of protein intrinsic t® the host, which is then cleaved off,, it has now been found that a segment composed of ©nly about TO amino acids ef fhe P~pr©tein from the trp operon of E« coli is particularly suitable for the formation of fusion proteins, specifically in th© region ef the sequence of arain© acids 23 te 23 CC» Yanofsfey et al», (Sucleic Acids Res,., 9 C1981!)S, also called IB®-pept ide hereinafter, Between the earfeosyl terminal end ef this peptide and ©f the amino acid sequence of the desired eukaryotic protein there is a sequence comprising ene or more genetically codable amino acids whieh permits the desired protein to be 20 cleaved off chemically or enzymatically- So preferred embodiments of this invention, the amino terminal end is followed Tay a short anin© aeid sequence composed of ILys-Ala, optionally followed by a sequence of 1 to HO, in particular .
H to 3, other genetically codable aain© acids, preferably 25 by two amino acids, in particular fey Lys-SlyiSence the invention relates to a fusion protein of the general formula Het-Xjj-D’-T-Z ’ in which n is zero or H, X ig a sequence of 1 t© 12 genetically codable amino aeids, preferably Lys-Ma, B* is a sequence of about 70 amino acids in the region „ of tb® sequence of amino acids 23-93 of the B-peptide in the trp operon of E. coli, „ ¥ denotes a sequence of one ®r mer® genetically codable amino aeids which permits the following amino acid sequence Z to b® cleaved off, and Z is a sequence of genetically codable amino acids» further aspects of the invention and preferred embodiments ar© described hereinafter and defined in the patent claims» Of course, it is advantageous if the undesired portion (intrinsic to th® host) of th® fusion protein is as small as possible since then the cell produces only little "ballast™ and hence th® yield ©f desired protein is high» Furthersore, when the undesired portion is cleaved off, fewer byproducts are produced, which facilitates working up» A factor (opposing this is that the (assumed^ ’’protective function™ of the undesired portion is to fee expected only above a (certain sis©» at has now ©merged, surprisingly, that the segment chosen according to the invention from the e-protein fulfils this task although it contains only about 70 amino acids.
In aany cases, ©specially in the preferred embodiment in which X represents Lys-Ala or contains this sequence at the W-terminal end, th® fusion protein formed is insoluble. Th® tatter can easily be separated from the soluble proteins, which greatly facilitates the working up and increases th© yield. The formation of an insoluble fusion c 30· protein is surprising since, on the on® hand, the bacterial portion of only about 70 amino acids is quit® snail and, . * on the other hand, it is a constituent of a protein which is present in solution in the host cell.
About 70 amino acids in the region of the sequence of asine acids 23 to 93 of the 0-peptide Eleans that it is possible to carry eut, in a manner known per se, variations, that is to say it is possible for individual an in© acids to b® deleted/· replaced or exchanged without this significantly changing the properties of th© fusion proteins according to the invention. The invention likewise relates to variations of this type.
The desired eukaryotic protein is preferably a biologically active protein/ such as a hirudin/ or a precursor of a pro110 tein of this typ©/ such as human proinsulin.
The fusion protein is tained by expression in a suitable US system, and in the particularly preferred embodiment is, after disruption of the host cell®/ isolated froa the sediment in which it is concentrated owing to its sparing solubility. Hence it is easy t® separate it fro® the soluble constituents of the cell.
Suitable host cells are all those for which expression systems are known , that is t© say naaaalian sells and saicr©©rganisnS/ preferably bacteria,, in particular E. eo>li sine©/ after all/ the bacterial portion of th© fusion protein is a protein intrinsic to the host E. soli.
Th® ©MA sequence which codes for the fusion protein according to the invention is incorporated/ in a known Banner/ into a vector which erasures satisfactory expression in the selected expression systen.
In bacterial, hosts, it is expedient to ehoos.e the promoter and operator from the group comprising Lae, .Tac, P^ ®r Pg of the phage a, hsp, ©Bp or a synthetic promoter, such as ar® described, for exanple, in ©erisan offenlegungs30 schrift 3/430/683 '(European Patent Application 0,1)73,1495.
A particularly suitable vector is one which contains the following elements ©f the trp operon ©f IE. colis the pro5 meter, th® operator and the ribosome binding sit® of th® L-peptide- Xt is particularly advantageous for the first three amino acids of this L-peptide to follow in the coding region, and then to be followed by a short amino acid sequence and th® amino acids 23 to 93 of th® ©-protein in th© trp operonTh® intermediate sequence V which sakes it possible to cleave ©ff the desired polypeptide depends on the composition ©f this desired peptides for example, if this con10 tains n© methionine it is possible for Ϋ to denote Het and then eheraical cleavage with cyanogen bromide is carried out» Xf ther© is cysteine at the carboxyl terminal end in the connecting member ¥, or if ¥ represents Cys, then cysteine-specific ensymatic cleavage or chemical eleav US age, for example after specific S-cyanylation, can be carried ©ut- if ther© is tryptophan at the carboxyl terminal end of th® bridging member ¥, or if ¥ represents Trp, then chemical cleavage with N-broraostaeeinimide ean b® carried out- Xf ¥ represents Asp-Pro, then proteolytic cleavage can be carried out in a manner known per s® <0- Piszkiewics et al», Biochemical and Biophysical Research Communications 40 <19705 11?3»-117S>. Th© Asp-Pro bond can, as has been found, be mad© even more acid-labile if Ϊ is B-Pr® or 6lw-lAsp5H-Pro, 2S rn denoting 1, 2 ©r 3» Jn these cases the cleavage products' obtained start at the N-terminal end with S>ro and terminate at the C-terminal end with Asp» Examples of enzymatic cleavages are likewise known, it also being possible to ose modified enzymes of improved specificity 291-2975» if th® desired eukaryotic peptide is human proinsulin if is possible to choose as the sequence ί a peptide sequence in which an anino acid which can be cleaved off by trypsin CArg, Lys) is bonded to the N-terminal 3S amino acid CPtoe) ©f the preinsulin, for example Ala-SerHet-Thr-Arg, sine® then the arginine-specific cleavage can be carried out with the protease trypsin. iff the desired protein do®® not contain the amino acid sequence Xle-SluGly-Arg, it is also possible to cleave with factor Xa CEuro5 peso Patent Application 0,. 161.937}, St is also possible in the design of sequence Y-to take ac- * count of the synthetic circumstances and to incorporate suitable cleavage sites for restriction enzymes. The hHA sequence corresponding to-the araino acid sequence Y can thus also assume the function of a linker or adapter.
Th© fusion protein according to the invention is advantageously expressed under the control of the trp operon of £. coli. A DHA segment containing the promoter and operator of th® trp operon is now commercially available. The ex15 pression of proteins under th® control off the trp operon has been described many times,, for example in European Application 0()36.776,. The induction off the trp ©peron can be effected by the absence ©f L-tryptophan and/or th© presence off indolyl-3-acrylic acid in the medium.
Under th® control off the trp operator there is ffirst transcripfcim of the DNA region. coding far the Irpeptide. This L-peptads «nidi is composed of 14 amino acids, contains L-tryptophan in each of the positions 10 and 11. The rate of ptoheln synthesis of the L-pepticls determines whether the downstream structural genes ar© Hfee25 wise translated or whether protein synthesis is terminated.
When there is a deficiency of L-tryptophan ther© now takes place slow synthesis ef the L-peptide as a result ®ff the low concentration of the tRNA for L-tryptophan, and the following proteins are synthesized. Sn contrast, when there 3t3 are high concentrat ions eff L-tryptophan the corresponding segment ©ff mRNA is rapidly read and termination ©ff protein biosynthesis takes place, since the nRHA assumes a terainatorlike structure The frequency of translation of a mRNA is greatly influenced by the nature of the nucleotides in ftbe vicinity of th® start codon» Thus,, on expression of a fusion protein with the aid of the trp operon, it appears favorable to insert th® nucleotides for th® first few amino acids of the Ltaeptid© for the start of th® structural gen® of the fusion protein® ϊη the preferred embodiment of the invention using the trp system the nucleotides of the first three amino acids of the L-peptide (called I·’-peptide hereinafter) were chosen ass. codons for the M-terminaX amino acids of the fusion protein» Hence th® invention also relates to vectors, preferably plasmids, for the expression of fusion proteins, the DBA of the vectors having the following features fro® the 5*~end Cin suitable order and in phase): a promoter, an operator, a ribosome binding sift© and the structural gen® for the fusion protein, the latter containing arain© acid seguence I (appendix) upstream of the seguence of th® desired protein» Upstream of the structural geo®, ©r as the first triplet of the structural gen®, there is located th® start codon CATS) and optionally further «©dons for ©ther amino acids which ar® arranged between th@ start codon and th® ©"-sequence or between th® P-sequence and the gen© for the desired protein» Th® choice of the sequence upstream of the structural gene depends en th® amino acid composition of th© desired protein, in order to make it possible to cleave the desired protein off from th© fusion protein.
It may prove advantageous in th® expression of the fusion protein according to the invention ft® modify individual triplets of the first few amino acids downstream of th® AT‘3 start codon in order t© prevent any base-pairing aft th® level ©f th® mRHA. Modifications of this type are, just as are modifications, deletions-or additions of individual amino acids in the P’-protein, familiar to those skilled in the art, and the invention likewise relates to them,.
S-Since relatively snail plasmids confer several advantages,? a preferred embodiment of the invention comprises the elimination of a PHA segment with th© structural gen® for tetraeyeline resistance from plasmid® derived from pSR 322«, It is advantageous t© delete th© segment from the Hindus restriction site at p©sition 2? to the Pvul2 restriction sit© at position 2066», It is particularly advantageous to delete a BHA segraent which is even somewhat larger from the plasmids according te the invention,?, by use ot the PvulI restriction site at the start (in the direction of reading) of the trp ©peron (which is located in a nonessential part),., It is thus possible to carry out direct ligation of the resulting] large fragment with the two Pvui restriction sites. The resulting ©I asn id,? which has been IS shortened by about 2 febp? effects an increase in expression? and this is possibly attributable to an increased e©py number in the host eel 1,.
The invention is illustrated in detail in the examples which a) Chromosomal E. cell BNA is cat with Mint I? and the 492 fragment which contains the promoter? operator? the structural gene of the L-peptide? the attenuator and the codons for the first six a®ino acids of the trp-Ε structural gene from the trp operon is isolated. This fragment is filled in with deoxynueleotide triphosphates with the aid of Klenow polymerase? linked at both end® to an oligonucleotide which contains a recognition site for Hindus and is then cut with Hindlll. Th© Hindlll fragment thus obtained is ligated into the Hindll! restriction site ©f pB8 322«, ptrpE2-1 (J.C. Edraann et a I.? is converted into the This results in the p Nature 291 (19815 5 pIas® i d p t rpL1 as d By use ' of th® synthetic oligonucleotides (B15 and ® C6A CAA TSA AA6 CAA A6G 3® CM1> 5° CCT TT© CTT TEA TT® T 3" (B2) which hybridise to the double- stranded oligo-- nucleotide Ή13Ϊ 5’ C@A CAA ISA AAG CAA AGG 3' 3 s T (STT ACT TTC STT TCC 5 s the BhIA sequence for the first three amino aeids of fch@ L-peptide is incorporated , and a restriction site (Stul for th® insertion of further BBA is formed, in the Clal site of the plasmid ptrpLI (figure 15. The plasmid ptrpLH is reacted with th© ensyra® Clal in accordance with th® manufacturer’s instructions, and the mixture is extracted with phenol and th® BMA is precipitated with ethanol. Th© opened plasmid is reacted with al fcaline phosphatase from E. coli to remove the phosphate groups at the S’-ends. The synthetic nucleotides are phosphorylated at the 5"-ends and ar® inserted, using T4 PHA ligase, into th© opened plasmid which has been treated with phosphatase. After th® ligase reaction is complete, transformation int© E. coli 294 and selection of th® transformants by Amp resistance and th® presence ©f a Stu2 restriction site are carried out.
About SOX ©f the resulting denes had the expected re25 striction site ? th® nucleotide sequence depicted in Figure 1 was confirmed by sequence analysis. The plasmid pH120/14 which contains downstream of the ribosome binding site for the L-peptide the nucleotide triplets for the first three amino acids of the L-peptide CL’-peptide), followed by a StuZ site which in turn permits the insertian of further ΕΊΗΙΑ and thus allows th® formation of fusion proteins having the first three amino acids of the L-peptide, is obtained. fo? The example of the oligonucleotide (MU’ employed above is used below to illustrate the chemical synthesis of such ©Iigonucleotides: Th® method of H.J- Sait et al., Nucleic Acids Research <1980? 11081-1090 is used to bond covalently the nucleoside at the 3’-end, that is to say guanosine In the present case, to a glass bead support (CFG («controlled pore glass? LCAA («long-chain alkylataine? supplied fey Pierce? via the 3’-hydroxyl group. This entails the guanosine feeing reacted as the H-2-isobutyryl 3'-θsuccinyl 5’-dimethoxytrityl ether with th® modified support in the presence of Η,Η’-dicyclohexylearfoodiimide and 4-dimethylarainopyridine, there being acylation of the amino radical of the long-chain amine ©n the support by the free carboxyl group of th© succinyl radical.
In the subsequent steps in the synthesis the base component is used as fhe dialkylamide ©r chloride of th© mono-methyl ester ef the 5*-0-di«eth©xytritylnucleoside20 ^’-phosphorous acid, th© adenine feeing in the form of the Νδ-feenzoyl compound, th® cytosine being in the form of the S4-bensoyl compound, th® guanine feeing in the form of fhe N2-is©fewtyryl compound, and th® thymine, which contains n© aain© group, feeing without a protectiv® group40 ® acetonitrile, f? 15 gan©l of the appropriate nucleoside phosphite and 1 ysaol of tetrazole in 0.3 ml of anhydrous acetonitrile ¢5 minutes), g) 201 acetic anhydride in tetrahydrofuran containing 401 lutidine and 101 dimethylarainopyridine <2 minutes), h) tetrahydrofuran, 1) tetrahydrofuran containing 201 water and 40® lutidine, j) 31 iodine in collidine/water/tetrahydrofuran in the ratio by volume 5:4:1, k) tetrahydrofuran and eethanol.
The terra phosphite in this context is defined as the monomethyl ©ster of th® deoxyribose-S’-raonoptoosphorous acid, the third valency being saturated by chlorine or a tertiary amino group, for example a diisopropylamino radical- The yields from the Individual steps in the synthesis can be determined after each detritylation reaction b) by spectrophotometry by raeasureraent of the absorption of the dimethoxytrityl cation at a wavelength ©f 496 in®» One® the synthesis ©f the oligonucleotide is complete, the methyl phosphate protective groups on the oligomer ar® cleaved off by us© of p-tlfoiocresol and triethylamine.
The oligonucleotide is then detached from the .solid support by treatraent with acapnia for 5 hours. Treatment of the oligomers with concentrated ammonia for 2 to 3 days quantitatively cleaves off th© amino protective groups en the bases. The crude product thus obtained is purified by high pressure liquid chromatography (HPLC) or by polyacrylamide gel electrophoresis.
The other oligonucleotides are also synthesised entirely correspondingly. c) The plasmid ptrpE5-1 CR. A. ftallewell et al., 'Sen® ? C19SO5 27-47) is reacted with the restriction enzymes Hindus and Sail in accordance with the manufacturer’s J 2 instructions, and the DMA fragment ®f about 620 bp is removed. Tb® synthetic oligonucleotides (M4> and (MS) ’ AGC TTC CAT GAC SC® T 3" CtU) 5" ACS CGT CAT GGA 5s CMS) 5 are phosphorylated, incubated together at 37®C and, by us® of DMA ligase, added ©nt© tb© blunt-ended DMA for proinsulin CH. Hetekam et al-, Sen® 19 0982) 1791835. After reaction with HindlO and SalX, the proinsulin DMA which has now been extended is covalently 10 incorporated into the opened plasmid using the enzyme T4 DMA ligase (Figure 2), this producing the plasmid pH106/4.
The plasmid pH106/4 is first reacted ©nee raore with Sail, the overlapping ends ar® filled in with Klenow polymerase to give blunt ends and the product is then incubated with the enzyme WstX- A DMA fragment of about 500 bp which contains the entire part coding for proinsulin and a segment of about 210 bp of the Bprotein from the trp ©peron of E. coli is isolated.
The DMA fragment is blunt-ended and is inserted into the StuS site of th® plasmid pH120/14, thus producing the plasmid pH154/25 (Figure'3>„ This is suitable for th© expression of a fusion protein under the control of the trp operon, in which th® amino acid sequence Ala-Ser-Het-Thr-Arg is located after the b’- and D’peptide anal is followed by the amino acid sequence of proinsulin.
Exampl© 2 The plasmid pH154/25 (Figure 3) is reacted with the re30 strictien enzymes BamE-Π and XmaXXX. The protruding ends are filled in with Klenow polymerase and linked using T4 DMA ligase. This results in the plasmid pM254 (figure -) which is suitable for the express ion . of a fusion protein hawing the amino acid sequence LJ, D’-proinsulin under the control of the trp promoter» The plasmid is somewhat smaller than pH154/25, which may be an advantage» S Example 3 Incubation ®f the plasmid pH254 (Example 2^ Figure 4) with the restriction enzymes Hlul and Sail is carried out to liberate a DNA segment of 280 bp, and this is removed™ Th© remainder of th® plasmid is converted with Klenow poly10 merase into th® blunt-ended form and is covalently cyclized again with DNA ligase» This results in th© plasmid pH255 (Figure 45 which is suitable for the insertion of a structural gene int© ©ne of the restriction sites HluS, Sail and EcoRI», The formation ©f a fusion protein with the L®, D’-protein is carried ©ut under inducing conditions» Of course, it is possible to insert further restriction sites inf© the plasmid pH255 by suitable linkers» Example 4 The plasmid pH154/25 (Figure 35 is incubated with the en20 zyraes HluX and EcoRI, and the liberated DNA fragment (about 300 bp) is removed. Th® remainder of th® plasmid is filled in with Klenow polymerase» Ring closure is effected by the action of DNA ligase» The resulting plasmid pH256 (Figure 55 ean be used for insertion of structural genes into th® EcoRI site» Example 5 Deletion of a 600 bp fragment from the plasmid p[-32Si> (Example 4; Figure 55 using the restriction enzymes BamHI and NruX results in the plasraid pH257 (Figure 55» For this purpose, pH256 is first incubated with BaraHI and blunt ends are generated with Klenow polymerase,., After incubation with NruX and removal of the 600 bp fragment th® formation of pH257 is effected after incubation with ©WA ligas©.
Example 6 Insertion of tb® lae repressor (P.J. Farabaugh? Nature 274? (1978) 765-76.9). into the .plasmid. pKK..177-3 CAuann et al,.,? Sene 25 (1983) 167) results in th® plasmid pJFUS. This is reacted with EcoRI and Sail? and th® remainder of the plasmid is isolated.
A fragment about 495 bp in six© is obtained from th© plasmid pH106/4 (Figure 2) by the action of Sail and incubation with Mstl.
The oligonucleotides CM6) and CH7) obtained by synthesis 5® ACS AAT TCA TSA AA6 CAA ASS 3® CH6) 5' CCT TTS CTT TCA ISA ATT cst 3® (N7) ar® phosphorylated and? us ing ΡΗΑ ligase? added onto fth® blunt-ended ©HA fragment. Reaction with EcoRI and Sail liberates overlapping ends which permit ligation into the opened plasmid pJF118.
After transformation int© E. soli 294 of the hybrid plasmid which ha® thus been obtained? th© correct clones ar® selected on the basis of the six© ®f the restriction fragments. This plasmid is called pJ120 (Figure 6).
The express ion of the fusion protein is carried ©ut in shaken flasks as follows: An overnight culture in lb medium Hiller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 19721*, containing 50 pg/ml ampicillln, of S- coli 294-transformants which contain the plasmid pJ120 is used to set up a fresh culture in the ratio of about Is 100, and the growth IS is followed by measurement of the Ob,., ^hen the Ob is 0.5, isopropyl S-B-galactopyranoside CXPT6) is added to the culture in an amount such that its concentration is 1 mH, and the bacteria ar© removed by centrifugation after 150 to 180 minutes. The bacteria are boiled for 5 minutes in a buffer mixture C7H urea, 0.12 SOS, 0.,1 W sodium phosphate, pH 7.0), and samples ar® applied to a SOS gel electrophoresis plate. After the electrophoresis, the bacteria which contain the plasmid pJ120 provide a protein band whieh corres10 ponds te the sise of the expected fusion protein and which reacts with antibodies against insulin. After the fusion protein has been isolated the expected proinsulin derivative can be liberated by cleavage with cyanogen bromide., After disruption of the bacteria (French Press; ( K s 0yroo-mi.il S and centrifugation, the L®, O’-proinsulin fusion protein is located in th® sediment, so that considerable amounts of the other proteins can row be removed with the supernatant.
Th® stated induction conditions apply to shake cultures; for larger fermentations it is advantageous to modify the OD values accordingly and, where appropriate, to vary the SPTG concentrations slightly.
Example 7 An overnight culture in LB medium containing 50 yg/cal ampi25 cillin is prepared from is. coli 294 transformants which contain the plasmid pH154/25 (Figure 5) and the next morning is diluted in the ratio of about 1:100 in 89 medium CJ. H„ Hiller, loc. cit.) containing 2000 pg/ml casamino acids and 1 pg/ml thiamine. When the OB s 0.5 indolyl-330 acrylic acid is added so that the final concentration is yg/nl. After incubation for 2 to 3 hours, th© bacteria ar® removed by centrifugation. SIBS gel electrophoresis shows a very pronounced protein band which is at the place.expected for the fusion protein and whieh reacts with anti35 bodies against insulin. After disruption ©f the bacteria and centrifugation, the L®, B’-proiwswlin fusion protein is located in the sediment so that, again, considerable amounts of the other proteins are now removed with the supernatant.
In the present case too, the stated induction conditions apply to shake-cultures.- Fermentations-in-larger- volumes require altered concentrations of easanino acids or addition of L-tryptophan.
Example 8 The plasmid pH154/25 (Figure 35 is opened with EcoRl, and ths protruding BBA single strands are filled in with Klenow polymerase. The DfiA thus obtained is incubated with the enzyme Mlu2, and th© PNA coding for insulin is cut out of the plasmid. Separation by gel electrophoresis is carried out to remove this fragment from the remainder of the plasmid, and th® remainder of the plasmid is isolated. shown in Figure 3 ©f German Offenlegungsschri' 3,429,430 (European Patent Application A1 0,171,0245 is reacted with the restriction enzymes Acc2 SaL and BNA fragment cont protrud been filled in with Kl the hirudin sequence is removed, f the Sail restriction site have polymerase, th® BHA segment is ligated with the synthetic OBA of th© formula (M85 5 CCC ACS Set C G y A h S Th r ACG T 0 3 * G S G TGC CCA TAC TCC A‘j Z*1 (to ffl e-i i i-a «3 <03 The ligation pros Suet is inc ubated with MluS. After the en- zyme has been inactivated a t SSUC, the Β NA mix sure is treated with bovine alkaline phosphatase at 37°C for on© hour.
Thi® is followed by removal of the phosphate®® and the restriction enzyme from the »ixtur© by extraction with phenol, and the Bid A is purified by ethanol precipitation. The BBA which'has thus been treated is inserted using T4 ligase ϊ·7 into the opened remainder of the plasmid pH154/25, this resulting in the plasmid pE<150 whieh has been characterized by restriction analysis and PHA sequencing by the method of Maxam and Gilbert (Figure 75Example 9 E. coli 294 bacteria which contain th© plasmid pK150 (Figure 7? are cultured in LB medium containing 30 to 50 yg/ml ampicillin at 37°C overnight» The culture is diluted in the ratio of 1:100 with H9 medium whieh contains 2000 pg/ml casamin© acids and 1 pg/al thiamin®, and the mixture is incubated at 37®C, mixing continuously» fe’hen the Qhjjggs 0.5 or 1, indotyl-3-acrylic acid is added to a final concentration of 15 pg/ml, and the mixture is incubated for 2 to 3 hours ©r 16 hours respectively. The bacteria are then removed by centrifugation and disrupted in 0.1 M sodium phosphate buffer CpH 6.5> under pressure. The sparingly soluble proteins are removed by centrifugation and analysed by SPS polyacrylamide gel electrophoresis. It emerges that cells whose trp operon has been induced con15 tain in the region below 20,003 Patterns but above 14,,000 ©aliens anew protein which is not found in «©«-induced sells. After the fusion protein has been isolated and reacted with cyanogen bromide hirudin is liberated.
Example 10 The constructs described hereinafter i®ermit th® introduction, upstream of the S’-erad ©f th® trp-© sequence, of ΘΝΑ sequences which contain as many recognition sites for various restriction enzymes as possible In order to incorporate the trp-D sequence into as many as possible ©f the wide variety ©f prokaryoctic expression systems.
The plasmids pUC12 and pUC13 CPharsaeia P-L Sioehemicals, 5401 St- Soars The Molecular Biology Catalogue 1983, Ap1 8 pendix, p. S9> contain a polylinker sequence, it being the intention to insert in the plasmid pUC13, between th® restric tion cleavage sites for Kraal and SacX, the Hstl-Hindlll .trp fragment from the plasmid p106/4 (FigureS) which is fused with the HindiΧΙ-hirudin-Sad fragment from th© plasmid pKISO (Figure 7).
For this purpose, the OHA of the plasmid pUd3 is first treated with the restriction enzyme Xaal. Th® ends of th® linearised plasmid ar© filled in by means of the Klenow polymerase reaction. After ©thanol precipitation, the BHA is treated with th® enzyae SasZ and is again precipitated from the reaction mixture with ©thanol. The ©MA is now reacted in an aqueous ligation mixture with the MstXHimdlll trp-P fragment isolated frost plasmid pHW6/4 and with th® HindllS-SaeS hirudin fragment isolated from th© plasmid pKISO, and T4 ©HA ligase.
Th© plasmid ρΚΙέΟ thus obtained now contains, immediately upstream of th® trp-B sequence, a multiple restriction enzyme recognition sequence which embraces restriction sites for the enzymes Xraal, SraaX, BamHl, Xbal, Hindi, Sail, Accl, Pstl and Hindlll. furthermore, an EcoRl restriction site is generated downstream ©f th© 3*-end of th© hirudin sequence in this construction (Figure 85» Example 11 Th© plasmid pHH51/5 is (as shown in the unpublished German Patent Application P 35 14 113.1, Example 1, Figure 15 prepared as follews: Th® plasmid ptrpLI (j„C. Edman et al., loc. cit) is opened with Clal and ligated with the synthe30 tically prepared, self-complementary oligonucleotide CH9) " pCSACCATSST 3’| «Μ95 The plasmid pH131/5 (Figure 9) thus obtained is opened at th© restriction sit®, which has been introduced in this Banner, for the restriction enzyme Ncol, and the resulting protruding single-stranded ends are filled in by means of the Klenow polymerase reaction» The linearized, blunt-ended DNA is now cut with th© enzyme EcoRX, and the larger of the two resulting DNA sequences is separated from the smal ler sequence by ethanol precipitation. The remainder of the plasmid DNA of the plasmid pH131/5 thus obtained is now ligated with a fragment from pKI&Q coding for trp-Dhirudin by reaction with T4 ligase. This fragment is cleaved out of the plasmid pK160 by opening th® plasmid with IHincXI and EcoRI. The fragment is removed from the remainder of the plasmid by gel electrophoresis, and is then eluted from th® gel material. The ligation product is transformed into Ε» coli Κ12» The clones containing plasmid DNA are isolated and characterized by restriction analysis and DNA sequence analysis- The plasmid pK170 thus obtained contains fused onto th© trp operator a DNA sequence which codes for Net-Asp-Ser-Arg-@ly-Ser-Pro-6lytrp-D’-Chirudin) (Figure 95« Example 12 The plasmid pJF11@ (Example 65 is opened with EcoRI, and the protruding DNA ends are converted into blunt ends by means of th® Klenow polymerase reaction. Th® DNA thus treated is then cut with th® enzyme Sail, and th® short EcoRX-SalX fragment is removed by gel electrophoresis» Th® plasmid pK 170 (Example 115 is cleaved with Ncol, and the protruding end® ar® converted int® blunt ends using Klenow .polymerase» The plasmid BNA is removed fro® the reaction mixture by ethanol precipitation and is treated with the enzymes MindXXS and BamHI. Two of the resulting fragments are isolated, namely the NcoX (with filled-in end5-trp-D"HindXSS fragment and the HindlXX-hirudin-BamHX fragraent (Serman ©ffenlegungsschrift 3,429,430)» The two fragments are isolated after separation by gel electro35 phoresis. Ο Sn addition, the iBamMI-SgilX-MirudinXX fragment shown in Figure 2 of German Offenlegungsschrift 3,429,430 is isolated. Xn a ligation reaction the four fragments, namely the remainder of th® plasmid pJF 118, the HcoS-trp-D HindSSS fragment, the HindlXl-hirudin-BarnHS fragment and - - the hi.rudinXX fragment, are now reacted., .togetherand the resulting plasmid pK 130 (Figure 10) is transformed into IE. coli K12-H 3110. Correct plasmids are shown by it being possible to detect an EcoRX-trp-B*-hirudin-SaIX fragment in the plasmid DMA. Th® trp-D’-hirudin sequence is now attached to the tac promoter. The fusion protein is expressed as in Example 6.
Example 13 Th® plasmids derived from pBR 322, such as pH120/1.4 (Ex15 ample 1, Figure 15, pH1S4Z25 (Example 1, Figure 3), pM256 (Example 4, Figure 5), pK150 (Example S, Figure 75 and pK17Q (Example 11, Figure 9), have - on the figures in th© clockwise direction - between the start codon of the fusion protein and the next Hindus sit® (corresponding to Hindus at position 29 in pBR322) an additional PvuIX site in the region of the fragment which contains the trp promoter and operator, but outside the promoter region.
St has now been found that by deletion of the DMA segment which is bounded by the PvuIX sit© described and th© Pvuis site which corresponds to position 2066 in pBR322, the yield of a cloned protein (or fusion protein) is distinctly increased .
The shortening of the plasmid pH154/25 to give pH154/23* is described by way of example hereinafter, it being pas30 sible for this to be effected correspondingly for the other plasmids mentioned above (the shortened plasmids likewise being identified by an asterisk); PH154/25 is reacted with PwuSS (in accordance with the nanu2 1 facturer's instructions), resulting in three fragmentss Fragment Is From the PvuII restriction site of the pr©insulin gene to the PvuXI restriction site corresponding to position 2064 in pBR322, Fragment 2s from the FvuX X restriction sit© near the trp promoter to the PvuII site of th© proinsulin gene and Fragment 3s from the FvuXl site near the trp promoter .10 fragment to th© PvuII sit® corresponding to position 2046 in pBR322« Th© fragments can be separated by electrophoresis on agaras and then isolated (Maniatis et al-, Molecular Cloning, Cold Spring Harbor, 1982).
Th© fragments 1 and £ ar® joined under blunt ©nd conditions 15 using the enzyme 14 ®NA ligase» -Transformation into E- col 294 is followed by testing for those colonies which contain a plasmid with the (complete proinsulin sequence and thus have th® fragments in th© desired order. pH 154/25* is depicted in Figure 11» A distinct increase in the proportion of observed on expression, which is carried in th® preceding examples.
Th® fusion protein is out as described Example 14 The plasmid pH 154/25* (Example 13, Figure 11) is digested 25 with Hindlll and Sail, and th® small fragment (having th® proinsulin sequence? is separated.©ff by gel electrophor©sis. Th© large fragment is isolated and ligated with the synthetic PNA l AI a / Trp Glu Asp Pro Met 11® Glu (Sly) CArg? 30 A SCT <^P (&> «’ab 6 AS SAT CCT ATS ATC GAS €G mo) ACC igt «Ο» oft luTt CTA SS A TAC m Ab CTC CCA SCT tfja «Πμ The plasmid pXnt13 (Figure 12) is produced.
The BHA (H10) codes for an amino acid sequence which contains several cleavage sites for chemical cleavages: a) Het for cyanogen bromide? ..b) Trp for N.-hroftosucc in im ide . (MBS ©r ®SI) c) Asp-Pro for proteolytic cleavage? the upstream ®lu additionally weakening th® Asp-Pro bond towards the action of acids.
The introduction of this HindXXX-SalX-linker (Ν10Ϊ into the 10 reading frame of a coded polypeptide thus allows th® options which have been mentioned for chemical cleavage off? depending on the amino acid sequence of the desired protein and on its sensitivity to the eleaving agents.
The figures are not to scale 3 Amino acid sequence I (23) Ser Asn Gly H i s Asn Val Val XI® Tyr 10 Arg Asn H i s Ile Pro Ala Sin Thr Leu lie 20 SI u Arg Leu Ala Thr Met Ser Asn Pro Val 30 Leu Met Leu Ser Pro Sly Pro Sly Val Pro 40 Ser Glu Ala Sly Cys Met Pro Glu Leu Leu 50 Thr Arg Leu Arg (a I y Lys Leu Pro Ile 11 e (SO Sly II® Cys Lew Sly His 6 In Ala lie Val 70 6I u <«, Ala sequence Ser AGC Asn AAT Glv • GGG Wi s j&> ή en ® Asn 1¾ « fs MAW Val nws Val GTG Φ TH β ATT ΰνίθ® oa Aj* I© ί^«Ρ Μ.Λ g^ Asn His tag _ 8 « £2> aa, «*· %·» Pro Mlfi Gln ^-ί%,0 CGC A A jSi%W a !Αφ» CCG RGG VWw l& S± ffl w » 20. ®>cp^i Leu TI«, st>e*i -β. Glu il«a« Leu ST ft ΪΡ ACC . TTA, όΛ Λ β w fpi WV7W TTG GCG ACC £0 at » 30 ® Met Ser Asn Pr© Val Leu Met Leu ATG AGT Λβφ at mMV! GTG CTG & ATG CTT &0 f£«O·»® «> A W- Gly Gly val ?ro Ser ejsgfT* CCT OGC CCC ββφ Use wl GTG CCG AGC 100 <® »1 Glu ft Gly 1 Cys Met Pro Glu Leu i^j 1¾ ffi Wi I’M!) 0 |^a e®»j m'UU iusfs^F* Vtei1 ATG Vi Vi Vs tT?, m .¾ VS £5.41¾ Ul CTC £0 Leu Thr Arg Leu Arg Gly Lys Leu CTC !« £*0 p> ά?5ι V# V# w(jt w TTG ViVa <& GGC AAG ΠΦΠ »si aa V") ISO (i? ¢(3 Fro -n lie Bly TTl fib Cys Leu Gly WWW w ATT 8*1*1» #% C-S» «Si tbuiVi % A«nm i£?& lA TGC C^C Wt f*9 «9? GGA 70 -531S GlH !»\*3 <»« -At, y fl flife «be*» ’W e>® "g ^3.4» Glu Ala Cat CAS BCG ΰΤώ βρ> <& GTC GAA GCT 2Θ0 (« ffl PATENT CLAIHSs
Claims (27)
1. A fusion protein of the formula Het~X n -©'-Y-Z in which m ' is xero or 1? X is a sequence of 1 to 12 genetically codable amino acids? ©* is a sequence ©f about 70 amino acids in th® region of the sequence of amin© acids 23-93 ©f the Θ-peptide in the trp operon of E. coli? Y denotes a sequence ©f one or more genetically codable amino acids which permits the following amino acid sequence 2 to fee cleaved off? and Z is a sequence of genetically codable amino acids.
2. ,., A fusion protein as claimed in claim *3? wherein n is on© and X eonprises 1 to 5 amino acids.
3. A fusion protein as claimed in claim 2? wherein Lys-Ala is located at the N-terainal end of X.
4. A fusion protein as claimed in ©ne or more of th® preceding claims? where in T contains at th® e-terminal end Met? Cys? Trp? Arg or Lys or on® of the groups a Pro or Slu-CAsp) B -Pr© er Xle-Slu-Sly-Arg? in which o denotes 1, 2 or 3, or consists of these amino acids or one of these groups.
5. A fusion protein as claimed im one or aore of the preceding claiss? wherein 1 denotes the amino acid sequence of human proimsulin ©r of a hirudin»
6. » A process for the preparation of the fusion proteins as claimed in ©lain® 1 t© 5? wherein a gene structure coding for these fusion proteins is expressed in a host cell? and the fusion protein is separated off»
7. The precess as claimed in claim 6? wherein the E>HA sequence ί (appendix) codes in the gene structure for ©·. S. The process as claimed in claim 6 or 7? wherein the feNA sequence (coding strand) S6 5* AAA GCA AAG S6C 3’ codes in the gene structure for X.
8. 9» Tine process as claimed in one or nor® of claims 6 to 8, wherein the gene structure is selected so that the fusion protein is insoluble.
9. 10» The process as claimed in one or more ©f claims 6 fc© 9, wherein the gen® structure is contained in phase in a vector which contains the promoter, the operator and the ribosome binding site of the L-peptide fro® the trp operon ©f £. coli.
10. 11. The process as claimed in claim 10, wherein the vector is a derivative pf p8R322, the segment from the HifidXXX site at position 29 to the PvuXI site at position 2006 having been deleted froa the p6R322 DNA.
11. 12. The process as claimed in ene or more of elairas 6 te 11, wherein the host cell is a bacterium.
12. 13. The process as clained in en® or nor® ef claims 6 t© 12, wherein the host cell is E. coli.
13. 14. A gene structure coding for fusion proteins as claimed in claims 1 t© 5.
14. 15. A vector containing a gene structure as claimed in elaiea 14.
15. 16. A derivative of the plasmid pBR322, the segment from the HindlXX site at position 29 t© the FvuSX site at position 206© having been deleted from the p3R522 DNA, containing a gene structure as claimed in dale 14w
16. 17» An expression system containing a vector as claimed in claim IS ©r 1&.
17. 18. £. coli containing a vector as claimed in claim 15 ©r 16.
18. 19. A process for the preparation ©f a eukaryotic protein, which coaprises cleavage ©ff, «heroically or enzymatically, of the amino acid sequence Z from a fusion protein as claised in claims 1 t© 5.
19. 20. Plasmids pH 154/25, pH 254, pH 255, pH 256, pH257, pH 120/14, pK 150, pK 160, pK 170, pK 180, pH 154/25*, pH 256*, pH 120/14*, pK 150*, pK 17©* and pXntl3.
20.
21. A fusion protein of the formula given and defined in claim 1, substantially as hereinbefore described and exemplified.
22. A process for the preparation of a fusion protein of 5 the formula given and defined in claim 1, substantially as hereinbefore described and exemplified.
23. A fusion protein of the formula given and defined in claim 1, whenever prepared by a process claimed in any one of claims 6 to 13 or 22. 10
24. A gene structure according to claim 14, substantially as hereinbefore described and exemplified.
25. A vector according to claim 15, substantially as hereinbefore described and exemplified.
26. A process according to claim 19 for the preparation 15 of a eukaryotic protein, substantially as hereinbefore described and exemplified.
27. A eukaryotic protein whenever prepared by a process claimed in claim 19 or 26. Dated this the 25th day of July, 1986 F. R. KELLY & CO. BY: > EXECUTIVE 27 Clyde Road, BaiCLsbridge, Dublin 4 AGENTS FOR THE APPLICANTS HOECHST AKTIENGESELLSCHAFT 11 Sheets Sheet 1 Met Lys Ala Lys GAAA'GCA'AAGB TGT TACH TCGTHCC L‘ CCTTTGCTTTCATTG LA A inn (3) AAfCAAACTAA, , F.R. KELLY & CO.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19853526995 DE3526995A1 (en) | 1985-07-27 | 1985-07-27 | FUSION PROTEINS, METHOD FOR THEIR PRODUCTION AND THEIR USE |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IE861977L IE861977L (en) | 1987-01-27 |
| IE59127B1 true IE59127B1 (en) | 1994-01-12 |
Family
ID=6276985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IE197786A IE59127B1 (en) | 1985-07-27 | 1986-07-25 | Fusion proteins, a process for their preparation oand their use |
Country Status (18)
| Country | Link |
|---|---|
| EP (1) | EP0211299B1 (en) |
| JP (1) | JPH07113040B2 (en) |
| KR (1) | KR950000299B1 (en) |
| AT (1) | ATE50596T1 (en) |
| AU (1) | AU595221B2 (en) |
| CA (1) | CA1336329C (en) |
| DE (2) | DE3526995A1 (en) |
| DK (1) | DK172618B1 (en) |
| ES (1) | ES2000278A6 (en) |
| FI (1) | FI86887C (en) |
| GR (1) | GR861957B (en) |
| HU (1) | HU197356B (en) |
| IE (1) | IE59127B1 (en) |
| IL (1) | IL79522A (en) |
| NO (1) | NO175640C (en) |
| NZ (1) | NZ216967A (en) |
| PT (1) | PT83065B (en) |
| ZA (1) | ZA865556B (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3738541A1 (en) * | 1987-11-13 | 1989-05-24 | Hoechst Ag | METHOD FOR INSULATING AND CLEANING HIRUDINE |
| DE3541856A1 (en) * | 1985-11-27 | 1987-06-04 | Hoechst Ag | EUKARYOTIC FUSION PROTEINS, THEIR PRODUCTION AND USE, AND MEANS FOR CARRYING OUT THE PROCESS |
| JPS62220192A (en) * | 1986-03-12 | 1987-09-28 | インタ−ナシヨナル・ミネラルズ・アンド・ケミカル・コ−ポレイシヨン | Hybrid phenotypic vector, its structure and use |
| AU1987088A (en) * | 1987-06-24 | 1989-01-19 | Novo Nordisk A/S | A process for preparing a protein or polypeptide, a dna sequence coding for the polypeptide, a microorganism containing the dna sequence as well as the polypeptide and its use as a pharmaceutical preparation |
| US5179196A (en) * | 1989-05-04 | 1993-01-12 | Sri International | Purification of proteins employing ctap-iii fusions |
| IL95495A (en) * | 1989-08-29 | 1996-10-16 | Hoechst Ag | Fusion proteins their preparation and use |
| US5358857A (en) * | 1989-08-29 | 1994-10-25 | The General Hospital Corp. | Method of preparing fusion proteins |
| US5227293A (en) * | 1989-08-29 | 1993-07-13 | The General Hospital Corporation | Fusion proteins, their preparation and use |
| GB8927722D0 (en) * | 1989-12-07 | 1990-02-07 | British Bio Technology | Proteins and nucleic acids |
| DE3942580A1 (en) * | 1989-12-22 | 1991-06-27 | Basf Ag | METHOD FOR PRODUCING HIRUDINE |
| PT821006E (en) | 1996-07-26 | 2004-09-30 | Aventis Pharma Gmbh | INSULIN DERIVATIVES WITH ENHANCED ZINC LINK |
| DE19726167B4 (en) | 1997-06-20 | 2008-01-24 | Sanofi-Aventis Deutschland Gmbh | Insulin, process for its preparation and pharmaceutical preparation containing it |
| DE19825447A1 (en) | 1998-06-06 | 1999-12-09 | Hoechst Marion Roussel De Gmbh | New insulin analogues with increased zinc formation |
| DE10114178A1 (en) | 2001-03-23 | 2002-10-10 | Aventis Pharma Gmbh | Zinc-free and low-zinc insulin preparations with improved stability |
| DE10227232A1 (en) | 2002-06-18 | 2004-01-15 | Aventis Pharma Deutschland Gmbh | Sour insulin preparations with improved stability |
| PL2201120T3 (en) | 2007-09-21 | 2014-05-30 | Cadila Healthcare Ltd | Gcsf fusion protein systems suitable for high expression of peptides |
| PT2349324T (en) | 2008-10-17 | 2017-12-06 | Sanofi Aventis Deutschland | Combination of an insulin and a glp-1 agonist |
| PL2498802T3 (en) | 2009-11-13 | 2015-06-30 | Sanofi Aventis Deutschland | Pharmaceutical composition comprising a glp-1 agonist, an insulin, and methionine |
| EP2498801B1 (en) | 2009-11-13 | 2018-01-24 | Sanofi-Aventis Deutschland GmbH | PHARMACEUTICAL COMPOSITION COMPRISING desPro36Exendin-4(1-39)-Lys6-NH2 AND METHIONINE |
| EP2611458B1 (en) | 2010-08-30 | 2016-09-21 | Sanofi-Aventis Deutschland GmbH | Use of ave0010 for the manufacture of a medicament for the treatment of diabetes mellitus type 2 |
| US9821032B2 (en) | 2011-05-13 | 2017-11-21 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical combination for improving glycemic control as add-on therapy to basal insulin |
| BR112014004726A2 (en) | 2011-08-29 | 2017-04-04 | Sanofi Aventis Deutschland | pharmaceutical combination for use in glycemic control in type 2 diabetes patients |
| AR087744A1 (en) | 2011-09-01 | 2014-04-16 | Sanofi Aventis Deutschland | PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OF A NEURODEGENERATIVE DISEASE |
| EP3229828B1 (en) | 2014-12-12 | 2023-04-05 | Sanofi-Aventis Deutschland GmbH | Insulin glargine/lixisenatide fixed ratio formulation |
| TWI748945B (en) | 2015-03-13 | 2021-12-11 | 德商賽諾菲阿凡提斯德意志有限公司 | Treatment type 2 diabetes mellitus patients |
| TW201705975A (en) | 2015-03-18 | 2017-02-16 | 賽諾菲阿凡提斯德意志有限公司 | Treatment of type 2 diabetes mellitus patients |
| KR20200080748A (en) | 2018-12-27 | 2020-07-07 | 주식회사 폴루스 | A Method for Purifying Proinsulin Using Anion Exchange Chromatography |
| KR20200080747A (en) | 2018-12-27 | 2020-07-07 | 주식회사 폴루스 | An Enzymatic Conversion Composition for Producing Insulin from Proinsulin and a Method for Producing Insulin from Proinsulin Using the Same |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GR68404B (en) * | 1979-06-01 | 1981-12-29 | Univ California | |
| ZA811368B (en) * | 1980-03-24 | 1982-04-28 | Genentech Inc | Bacterial polypedtide expression employing tryptophan promoter-operator |
| WO1986005513A1 (en) * | 1985-03-18 | 1986-09-25 | Gene Labs, Inc. | Hybrid-gene cassette vector |
| GB8507666D0 (en) * | 1985-03-25 | 1985-05-01 | Wellcome Found | Epidermal growth factor production |
| WO1986006099A1 (en) * | 1985-04-08 | 1986-10-23 | Genetic Systems Corporation | EXPRESSION AND DIAGNOSTIC USE OF gag ENCODED PEPTIDES WHICH ARE IMMUNOLOGICALLY REACTIVE WITH ANTIBODIES TO LAV |
-
1985
- 1985-07-27 DE DE19853526995 patent/DE3526995A1/en not_active Withdrawn
-
1986
- 1986-07-19 DE DE8686109945T patent/DE3669175D1/en not_active Expired - Lifetime
- 1986-07-19 AT AT86109945T patent/ATE50596T1/en active
- 1986-07-19 EP EP86109945A patent/EP0211299B1/en not_active Expired - Lifetime
- 1986-07-24 FI FI863041A patent/FI86887C/en not_active IP Right Cessation
- 1986-07-24 ES ES8600544A patent/ES2000278A6/en not_active Expired
- 1986-07-25 GR GR861957A patent/GR861957B/en unknown
- 1986-07-25 PT PT83065A patent/PT83065B/en unknown
- 1986-07-25 DK DK198603554A patent/DK172618B1/en not_active IP Right Cessation
- 1986-07-25 NO NO863000A patent/NO175640C/en unknown
- 1986-07-25 ZA ZA865556A patent/ZA865556B/en unknown
- 1986-07-25 AU AU60565/86A patent/AU595221B2/en not_active Expired
- 1986-07-25 NZ NZ216967A patent/NZ216967A/en unknown
- 1986-07-25 IL IL79522A patent/IL79522A/en not_active IP Right Cessation
- 1986-07-25 CA CA000514682A patent/CA1336329C/en not_active Expired - Lifetime
- 1986-07-25 HU HU863100A patent/HU197356B/en unknown
- 1986-07-25 IE IE197786A patent/IE59127B1/en not_active IP Right Cessation
- 1986-07-26 JP JP61176558A patent/JPH07113040B2/en not_active Expired - Lifetime
- 1986-07-26 KR KR1019860006142A patent/KR950000299B1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| AU595221B2 (en) | 1990-03-29 |
| NO175640B (en) | 1994-08-01 |
| JPS6229600A (en) | 1987-02-07 |
| FI86887B (en) | 1992-07-15 |
| KR950000299B1 (en) | 1995-01-13 |
| PT83065B (en) | 1989-02-28 |
| IL79522A0 (en) | 1986-10-31 |
| ES2000278A6 (en) | 1988-02-01 |
| HUT43629A (en) | 1987-11-30 |
| IL79522A (en) | 1991-08-16 |
| HU197356B (en) | 1989-03-28 |
| EP0211299A3 (en) | 1988-06-01 |
| AU6056586A (en) | 1987-01-29 |
| DK355486A (en) | 1987-01-28 |
| EP0211299B1 (en) | 1990-02-28 |
| DE3526995A1 (en) | 1987-02-05 |
| ZA865556B (en) | 1987-03-25 |
| DK355486D0 (en) | 1986-07-25 |
| FI863041A0 (en) | 1986-07-24 |
| KR870001312A (en) | 1987-03-13 |
| NO863000L (en) | 1987-01-28 |
| FI86887C (en) | 1992-10-26 |
| IE861977L (en) | 1987-01-27 |
| FI863041A (en) | 1987-01-28 |
| EP0211299A2 (en) | 1987-02-25 |
| PT83065A (en) | 1986-08-01 |
| GR861957B (en) | 1986-11-25 |
| ATE50596T1 (en) | 1990-03-15 |
| NO863000D0 (en) | 1986-07-25 |
| DE3669175D1 (en) | 1990-04-05 |
| CA1336329C (en) | 1995-07-18 |
| NO175640C (en) | 1994-11-09 |
| DK172618B1 (en) | 1999-03-01 |
| NZ216967A (en) | 1988-08-30 |
| JPH07113040B2 (en) | 1995-12-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MK9A | Patent expired |