DE2922496A1 - Protein cpds. e.g. hormones isolation from fusion proteins - by selective enzymatic hydrolysis of tetra:peptide sequences contg. proline and glycine - Google Patents

Protein cpds. e.g. hormones isolation from fusion proteins - by selective enzymatic hydrolysis of tetra:peptide sequences contg. proline and glycine

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DE2922496A1
DE2922496A1 DE19792922496 DE2922496A DE2922496A1 DE 2922496 A1 DE2922496 A1 DE 2922496A1 DE 19792922496 DE19792922496 DE 19792922496 DE 2922496 A DE2922496 A DE 2922496A DE 2922496 A1 DE2922496 A1 DE 2922496A1
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protein
proline
pro
gly
sequence
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Joachim Dr Daum
Hartmut Prof Dr Seliger
Gerhard Dr Siewert
Michael Dr Toepert
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Bayer Pharma AG
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Schering AG
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Priority to DE19792922496 priority Critical patent/DE2922496A1/en
Priority to IL60184A priority patent/IL60184A/en
Priority to JP7158880A priority patent/JPS5668399A/en
Priority to EP80730038A priority patent/EP0020290B1/en
Priority to DE8080730038T priority patent/DE3068508D1/en
Priority to AT80730038T priority patent/ATE8411T1/en
Publication of DE2922496A1 publication Critical patent/DE2922496A1/en
Priority to US06/395,433 priority patent/US4543329A/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • C07K14/57554—Prolactin
    • 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
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

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  • Endocrinology (AREA)
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  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

In a new process for the prodn. of proteins, the C-terminal of a protein contg. the tetrapeptide sequence Pro-Xyz-Gly-Pro (where Xyz represents any amino acid) is enzymatically cleaved to the protein sequence following the tetrapeptide sequence. Sepn. of foreign protein residues (e.g. mammalian proteo-hromones) from microbial protein residues in fusion proteins produced by genetically modified microorganisms. The enzymatic hydrolysis can be made specific for the defined tetrapeptide residue. Unlike the known cyanogen bromide method, it does not atack methonine residues.

Description

Die vorliegende Erfindung besteht in einem Verfahren zur HerstellungThe present invention resides in a method of manufacture

von Proteinen, das dadurch gekennzeichnet ist, daß man von einem Protein, das die Tetrapeptidsequenz Pro-Xyz-Gly-Pro enthält, wobei Xyz jede beliebige Aminosäure bedeuten kann, enzymatisch die C-terminal auf die Tetrapeptidsequenz folgende Proteinsequenz abspaltet.of proteins, which is characterized in that one of a protein, which contains the tetrapeptide sequence Pro-Xyz-Gly-Pro, where Xyz is any amino acid can mean enzymatically the protein sequence following the tetrapeptide sequence C-terminally splits off.

Bei der Synthese von Fremdproteinen (z.B. Säugetier-Proteohormone) durch genetisch modifizierte Mikroorganismen wird das Fremdgen, das die gewünschte Proteinsequenz codiert, in ein Strukturgen des Mikroorganismus eingebaut. Dadurch bleiben die Pegulationsorte auf der mikrobiellen DNA funktionsfähig und die Proteinbiosynthese kann in üblicher Weise ablaufen. Das Fremden, das am 3'-Ende des codogenen Stranges ein oder mehrere Stopcodons enthält, kann durch Synthese oder über Isolierung der mRNA und reverse Transkription als cDNA erhalten werden. Als Primärprodukt der Proteinsynthese wird dann ein Fusionspr otein erhalten1 das aminoterminal eine mehr oder weniger lange Sequenz des mikrobiellen Proteins und carboxylterminal das gewünschte Fremdprotein enthält. Aus diesem Fusionsprotein muß durch eine proteinchemische Reaktion das Fremdprotein abgespalten werden. Die einzige für diese Spaltung zur Zeit bekannte Methode ist die Reaktion mit Bromcyan, die zu einer Spaltung der Peptidsequenz am Carboxylende von Methionin-Resten führt (S.B.In the synthesis of foreign proteins (e.g. mammalian proteohormones) through genetically modified microorganisms, the foreign gene becomes the desired one Coded protein sequence, incorporated into a structural gene of the microorganism. Through this the pegulation sites on the microbial DNA and protein biosynthesis remain functional can run in the usual way. The alien that is at the 3 'end of the codogenic strand Contains one or more stop codons can be synthesized or isolated by the mRNA and reverse transcription can be obtained as cDNA. As the primary product of protein synthesis a fusion protein is then obtained1 the amino terminal one more or less long sequence of the microbial protein and carboxyl terminal the desired foreign protein contains. From this fusion protein, the Foreign protein are split off. The only one currently known for this split The method is the reaction with cyanogen bromide, which leads to a cleavage of the peptide sequence on Carboxyl end of methionine residues leads (S.B.

Needleman,protein sequence determination, Springer Verlag, 1970, N.Y.). Dazu ist erforderlich, daß das Fremdgen am 5'-Ende des codogenen Stranges ein zusätzliches Cm dorn für Methionin enthält. Dieses Verfahren versagt jedoch, wenn in dem gewünschten Fremdprotein weitere Methionin-Reste vorkommen. Zusätzlich hat die Bromcyanspaltung den Nachteil, an verschiedenen anderen Aminosäuren zu Nebeereaktionen zu führen.Needleman, protein sequence determination, Springer Verlag, 1970, N.Y.). This requires that the foreign gene has an additional one at the 5 'end of the codogenic strand Contains cm thorn for methionine. However, this method fails when in the desired manner Foreign protein further methionine residues occur. In addition, the cyanogen bromide cleavage the disadvantage of leading to negative reactions with various other amino acids.

Es wurde nun gefunden, daß eine Gewinnung des Fremdproteins ohne übliche Nebenreaktionen auch in Gegenwart von Methionin-Resten durch eine selektive enzymatische Hydrolyse gelingt, wenn in dem Fusionsprotein am aminoterminalen Ende der Fremdprotein-Sequenz ein Tetrapeptid der allgemeinen Formel Pro-Xyz-Gly-Pro, wobei Xyz jede beliebige Aminosäure sein kann, steht. Zunächst wird selektiv die Xyz-Gly-Bindung mit einer Collagenase (E.C. 3.4.24.3.) gespalten und anschließend der Glycin-Rest mit einer Aminoacylprolinaminopeptidase (Aminopeptidase-P,E.C. 3.4.11.9.) und der Prolin-Rest mit einer Prolinaminopeptidase (E.C. 3.4.11.5.) entfernt.It has now been found that recovery of the foreign protein without the usual Side reactions also in the presence of methionine residues by a selective enzymatic Hydrolysis succeeds when in the fusion protein at the amino terminal end of the foreign protein sequence a tetrapeptide of the general formula Pro-Xyz-Gly-Pro, where Xyz is any Can be amino acid, stands. First, the Xyz-Gly bond with a Collagenase (E.C. 3.4.24.3.) Cleaved and then the glycine residue with a Aminoacylproline aminopeptidase (aminopeptidase-P, E.C. 3.4.11.9.) And the proline residue removed with a proline aminopeptidase (E.C. 3.4.11.5.).

Das erfindungsgemäße Verfahren besteht daher aus der spezifischen enzymatischen Spaltung eines, durch Genetic Engineering erhaltenen Fusionsprotein, das an der Verknüpfungsstelle von originaler Proteinsequenz und Fremdprot ein eine Collagenase-spezifische Tetrapeptidsequenz der allgemeinen Formel Pro-Xyz-Gly-Pro hat, wobei für die enzymatische Spaltung die Enzyme Collagenase, Aminoacylprolin aminopeptidase und Prolinaminopeptidase verwendet werden.The method according to the invention therefore consists of the specific enzymatic cleavage of a fusion protein obtained by genetic engineering, the one at the junction of the original protein sequence and foreign protein Collagenase-specific tetrapeptide sequence of the general formula Pro-Xyz-Gly-Pro has, whereby for the enzymatic cleavage the enzymes collagenase, aminoacylproline aminopeptidase and proline aminopeptidase can be used.

Unter Xyz versteht man alle natürlich vorkommenden Aminosäuren, die im genetischen Code enthalten sind.Xyz is understood to mean all naturally occurring amino acids that contained in the genetic code.

Die für die enzymatische Abspaltung des gewünschten Proteins aus dem Fusionsprotein erforderliche Collagenase muß weitgehend frei von anderen Proteasen sein. Das Enzym wird z.B. von Clostridium histolyticum produziert und kann durch Fermentation in üblicher Weise nach bekannten Verfahren gewonnen werden. Es ist in gereinigter Form auch kommerziell erhältlich. Zur Blockierung der in den meisten Präparationen noch in geringer Menge enthaltenen Proteasen wird bei der Inkubation mit dem Fusionsprotein ein Proteaseinhi-hit:or z.B. Dllsopropyllfuorphosphat augesetzt. Collagenase selbst wird durch diesen Inhibitor nicht gehemmt. Bei dieser Umsetzung entsteht das um die Sequenz Gly-Pro aminoterminal verlängerte gewünschte Fremdprotein.For the enzymatic cleavage of the desired protein from the The collagenase required for fusion protein must be largely free of other proteases be. The enzyme is e.g. produced by Clostridium histolyticum and can be through Fermentation can be obtained in the usual way by known methods. It is also commercially available in purified form. To block in most Preparations still contained in small amounts of proteases is used during the incubation with the fusion protein a protease protein hit: or e.g. isopropyl fluorophosphate added. Collagenase itself is not inhibited by this inhibitor. In this implementation the desired foreign protein is formed which has been extended by the sequence Gly-Pro aminoterminally.

Es kann aus dem Reaktionsgemisch abgetrennt werden. Die dafür einzusetzenden Methoden richten sic nach den speziellen Eigenschaften der Verbindung, die im wesentlichen von dem gewünschten Fremdprotein (z.B. Proinsulin, Insulin A- oder ß-Kette, ACTH, STH) bestimmt werden.It can be separated from the reaction mixture. The ones to be used for this Methods are based on the special properties of the compound, which are essentially of the desired foreign protein (e.g. proinsulin, insulin A or ß-chain, ACTH, STH) can be determined.

Die Abspaltung des Glycin- und des Prolin-Restes mit den beiden spezifischen Aminopeptidasen kann in zwei getrennten Hydrolyseansätzen nacheinander oder wie in den meisten Fällen auch durch gemeinsame Inkubation mit beiden Enzymen erreicht werden. Die Aminopeptidase-P kann aus einem Escherichia coli-Stamm, z.B. dem Stamm B wie in Biochem. and Biophys. Res. Comm. Vol. 32, 658-663 (1968) beschrieben durch Ammonsulfat-Faktionierung, Aceton-Fällung sowie durch Chromatographie an Calciumphosphat, DEAE-Cellulose und Sephadex G-200 isoliert werden. Die Prolinaminopeptidase wird ebenfalls aus einem Escherichia coli-Stamm, z.B. aus dem Stamm K 12 isoliert. Sie wird nach Ammonsulfat- Fraktionierung und zweimaliger Chromatographie an DEAE-Cellulose ausreichend rein erhalten. Sie muß frei von weniger spezifischen Aminopeptidase sein tJ.Biol.The cleavage of the glycine and proline residues with the two specific ones Aminopeptidases can be used in two separate hydrolysis batches one after the other or as in most cases also achieved by joint incubation with both enzymes will. The aminopeptidase-P can be obtained from an Escherichia coli strain such as the strain B as in Biochem. and Biophys. Res. Comm. Vol. 32, 658-663 (1968) described by Ammonium sulphate fractionation, acetone precipitation and chromatography on calcium phosphate, DEAE cellulose and Sephadex G-200 can be isolated. The proline aminopeptidase will also from an Escherichia coli strain, e.g. isolated from the K 12 strain. she becomes after ammonium sulphate fractionation and chromatography twice on DEAE cellulose get sufficiently pure. It must be free of less specific aminopeptidase his tJ.Biol.

Chem. 294, 1740-1746 (1959); ibid. 237, 2207-2212 (1962)J.Chem. 294, 1740-1746 (1959); ibid. 237: 2207-2212 (1962) J.

Das in freier Form vorliegende gewünschte Fremdprotein wird nach den üblichen Methoden der Proteinreinigung in reiner Form isoliert.The desired foreign protein present in free form is after the common methods of protein purification isolated in pure form.

Die dabei eingesetzten Verfahren werden von den Eigenschaften des Fremdproteins (z.B. Proinsulin, Insulin A- oder ß-Kette, ACTH, STH) und den n Eigenschaften der abzutrennenden. Verbindungen bestimmt.The methods used are dependent on the properties of the Foreign protein (e.g. proinsulin, insulin A or ß-chain, ACTH, STH) and the n properties the to be separated. Connections determined.

Beispiel 1 140 mg Z-Gly-Pro-Leu-Gly-Pro-Insulin-A-Kette (50 ymol) werden in 10 ml 0,5 mol/l Tris-Puffer (pH 7,2) gelöst, der 0,1 mol/l Calcium-Acetat und zur Verhinderung einer Hydrolyse an unerwünschter Stelle durch Verunreinigung der Collagenase durch andere Proteasen 0,1 mmol/l Diisopropylfluorphosphat enthält, und mit 10 mg Collagenase versetzt. Nach 60 min bei 280C wird die Reaktion durch Zugabe von Athanol gestoppt. Nach dem Abfiltrieren von ausgefälltem Enzym wird der Alkohol im Vakuum weitgehend abgedampft und die Lösung durch Ghromatographie an Sephadex G-15 mit 0,01 mol/l Ammonium hydrogencarbonat als Elutionsmittel entsalzt. Das Eluat wird im Vakuum auf ein kleines Volumen eingeengt und gefriergetrocknet.Example 1 140 mg Z-Gly-Pro-Leu-Gly-Pro-Insulin A chain (50 μmol) are dissolved in 10 ml of 0.5 mol / l Tris buffer (pH 7.2), the 0.1 mol / l calcium acetate and to prevent hydrolysis in undesirable locations due to contamination the collagenase contains 0.1 mmol / l diisopropyl fluorophosphate due to other proteases, and mixed with 10 mg collagenase. After 60 min at 280C the reaction is complete Addition of ethanol stopped. After the precipitated enzyme has been filtered off, the Alcohol largely evaporated in vacuo and the solution by chromatography Sephadex G-15 is desalinated with 0.01 mol / l ammonium hydrogen carbonate as the eluent. The eluate is concentrated to a small volume in vacuo and freeze-dried.

Ausbeute 110 mg Gly-Pro-Insulin-A-Kette. Mittels Endgruppenbestimmung (DNP-Methode) kann Glycin als N-terminale Aminosäure nachgewiesen werden. Das erhaltene Produkt wird direkt zur weiteren Umsetzung eingesetzt.Yield 110 mg Gly-Pro-Insulin A chain. By means of end group determination (DNP method), glycine can be detected as an N-terminal amino acid. The received Product is used directly for further implementation.

Beispiel 2 Getrennte Abspaltung der ersten beiden N-terminalen Aminosäuren 50 mg Gly-Pro-Insulin-A-Kette werden in 1 ml 0,1 mol/l Tris-Puffer (pH 8,5), der 0,5 mmol/1 MnCl2 enthält, gelöst. 2 µg Aminopeptidase-P werden zugegeben und 1 h bei 37°C inkubiert. Durch kurzes Erhitzen auf 600C wird inaktiviert und ohne weitere Reinigung 20 g Prolin-xminopeptidase zugegeben. Nach 24 h bei 37°C werden die hochmolekularen Proteine durch Ausfällen mit Filkohol und Filtrieren entfernt. Das Filtrat wird im Vakuum eingeengt, in 0,01 mol/l Ammonium hydrogencarbonat-Puffer (pH 9), aufgenommen und durch Chromatographie an Sephadex G-25 entsalzt. Die Insulin-A-Ketten-Fraktion wird direkt auf eine AE-Cellulose-Säule aufgebracht und mit einem Ammoniumhydrogencarbonat-Gradienten (0,01-0,1 mol/ , pH 9) chromatographiert. Durch Einengen und Gefriertrocknen der Insulin-A-Ketten-Fra'Stion werden 40 mg Insulin-A-Kette erhalten Aminosäureanalyse: ber. Asp2.00Thr1.00Ser2.00Glu4.00Gly1.00Cys4.00Val1.00Ile2.00Leu2.00Tyr2.00 gef. Asp1.98Thr0.92Ser1.64Glu3.79Gly1.02Cysn.b.Val0.98Ile1.99Leu2.04Tyr1.86 Beispiel 3 Gemeinsame Abspaltung der ersten beiden N-terminalen-Aminosäuren Wie in Beispiel 2 werden 50 mg Gly-Pro-Insulin-A-Kette in 1 ml Tris-Puffer glöst. Anschließend werden 2 Mg Aminopeptidase-P und 20 µg Proliniminopeptidase zugesetzt. Nach einer Reaktionszeit von 18 h bei 37°C wird mit Alkohol gefällt und wie im Beispiel 2 aufgearbeitet.Example 2 Separate cleavage of the first two N-terminal amino acids 50 mg Gly-Pro-Insulin-A-chain are in 1 ml 0.1 mol / l Tris buffer (pH 8.5), the Contains 0.5 mmol / 1 MnCl2, dissolved. 2 ug aminopeptidase-P are added and 1 h incubated at 37 ° C. Brief heating to 600C inactivates and without any further Purification 20 g of proline xminopeptidase were added. After 24 h at 37 ° C, the high molecular weight Proteins removed by precipitation with alcohol and filtration. The filtrate will concentrated in vacuo, taken up in 0.01 mol / l ammonium hydrogen carbonate buffer (pH 9) and desalted by chromatography on Sephadex G-25. The insulin A chain fraction is applied directly to an AE cellulose column and with an ammonium hydrogen carbonate gradient (0.01-0.1 mol /, pH 9). By concentrating and freeze-drying the Insulin A chain Fra'Stion, 40 mg of insulin A chain are obtained. ber. Asp2.00Thr1.00Ser2.00Glu4.00Gly1.00Cys4.00Val1.00Ile2.00Leu2.00Tyr2.00 found. Asp1.98Thr0.92Ser1.64Glu3.79Gly1.02Cysn.b.Val0.98Ile1.99Leu2.04Tyr1.86 example 3 Joint cleavage of the first two N-terminal amino acids As in the example 2, 50 mg Gly-Pro-Insulin-A-chain are dissolved in 1 ml Tris buffer. Then be 2 mg aminopeptidase-P and 20 µg proline iminopeptidase were added. After a reaction time of 18 h at 37 ° C. is precipitated with alcohol and worked up as in Example 2.

Ausbeute 30 mg Insulin-A-Kette.Yield 30 mg of insulin A chain.

Beispiel 4 200 mg Z-Gly-Pro-Leu-Gly-Pro-Insulin-B-Kette werden wie in Beispiel 1 umgesetzt.Example 4 200 mg of Z-Gly-Pro-Leu-Gly-Pro-Insulin B chain are like implemented in example 1.

Ausbeute 150 mg an Gly-Pro-Insulin-B-Kette, die ohne weitere Reinigung weiter vorarbeitet werden.Yield 150 mg of Gly-Pro-Insulin B-chain without further purification further preparatory work.

Beispiel 5 100 mg nach Beispiel 4 hergestellte Gly-»ro-Insulin-B-Kette werden in 5 ml Trispuffer gelöst und wie in Beispiel 2 nacheinander mit Aminopeptidase-P und Prolinaminopeptidase inkubiert, an Sephade G-25 entsalzt und durch Chromatographie gereinigt.Example 5 100 mg Gly- »ro-insulin B-chain produced according to Example 4 are dissolved in 5 ml of Tris buffer and sequentially with aminopeptidase-P as in Example 2 and proline aminopeptidase, desalted on Sephade G-25 and chromatographed cleaned.

Ausbeute 70 mg Insulin-B-Kette.Yield 70 mg insulin B chain.

Aminosäurenanalyse: ber.: Asp1.00Thr1.00Ser1.00Glu3.00Gly3.00Pro1.0Cys2.00Phe3.00Val3.00 Leu4.00Ala2.00Tyr2.00His2.00Lys1.00Arg1.00 gef.: Asp0.95Thr0.92Ser0.83Glu3.12Gly3.02Pro0.98Cys1.75Phe2.98Val3.10 Leu3.96Ala2.04Tyr1.89His1.94Lys1.06Arg0.97 Beispiel 6 15 mg Z-Gly-Pro-Gly-Gly-Pro-Ala-Met-Glu-His-Phe-Arg-Trp-Gly werden wie in Beispiel 1 umgesetzt.Amino acid analysis: calculated: Asp1.00Thr1.00Ser1.00Glu3.00Gly3.00Pro1.0Cys2.00Phe3.00Val3.00 Leu4.00Ala2.00Tyr2.00His2.00Lys1.00Arg1.00 found .: Asp0.95Thr0.92Ser0.83Glu3.12Gly3.02Pro0.98Cys1.75Phe2.98Val3.10 Leu3.96Ala2.04Tyr1.89His1.94Lys1.06Arg0.97 Example 6 15 mg Z-Gly-Pro-Gly-Gly-Pro-Ala-Met-Glu-His-Phe-Arg-Trp-Gly are implemented as in Example 1.

Ausbeute 10 mg Gly-Pro-Ala-Met-Glu-His-Phe-Arg-Trp-Gly.Yield 10 mg Gly-Pro-Ala-Met-Glu-His-Phe-Arg-Trp-Gly.

Beispiel 7 10 mg nach Beispiel 6 hergestelltes Gly-Pro-Ala-Met-Glu-His-Phe-Arg-Trp-Gly werden wie in Beispiel 3 mit Aminopeptidase-P und Prolinaminopeptidase gemeinsam umgesetzt. Zur Reinigung wird nach dem Ausfällen der Enzyme mit Äthanol auf ein kleines Volumen eingeengt und über Carboxymethylcellulose mit einem Ammoniumacetat-Gradienten (0,01 bis 0,2 mol/l)chromatographiert.Example 7 10 mg of Gly-Pro-Ala-Met-Glu-His-Phe-Arg-Trp-Gly prepared according to Example 6 are as in Example 3 with aminopeptidase-P and proline aminopeptidase together implemented. After the enzymes have precipitated, they are cleaned with ethanol Small volume and concentrated over carboxymethyl cellulose with an ammonium acetate gradient (0.01 to 0.2 mol / l) chromatographed.

Ausbeute 7 mg Ala-Met-Glu-His-Phe-Arg-Trp-Gly.Yield 7 mg of Ala-Met-Glu-His-Phe-Arg-Trp-Gly.

Aminosäureanalyse: ber. Glu1.00Gly1.00Phe1.00Ala1.00Met1.00His1.00Arg1.00Trp1.00 gef. Glu1.03Gly1.01Phe0.98Ala1.02Met0.81His0.93Arg0.97Trpn.b.Amino acid analysis: calculated Glu1.00Gly1.00Phe1.00Ala1.00Met1.00His1.00Arg1.00Trp1.00 found Glu1.03Gly1.01Phe0.98Ala1.02Met0.81His0.93Arg0.97Trpn.b.

Claims (3)

Verfahren zur spezifischen Abspaltung von Proteinsequenzen aus Proteinen Patentansprüche 1) Verfahren zur Herstellung von Proteinen, dadurch gekennzeichnet, daß man von einem Protein,das die Tetrapeptidsequenz Pro-Xyz-Gly-Pro enthält, wobei Xyz jede beliebige Aminosäure bedeuten kann, enzymatisch die C-terminal auf die Tetrapeptidsequenz folgende Proteinsequenz abspaltet.Process for the specific cleavage of protein sequences from proteins Claims 1) Process for the production of proteins, characterized in that that one of a protein which contains the tetrapeptide sequence Pro-Xyz-Gly-Pro, wherein Xyz can mean any amino acid that is enzymatically linked to the C-terminal Tetrapeptide sequence cleaves the following protein sequence. 2) Verfahren zur Herstellung von Proteinen nach Anspruch 1, dadurch gekennzeichnet, daß man die Xyz-Gly-Bindung in der Tetrapertidsequenz mit einer Collagenase (E.C.3.4.24.3.) selektiv spaltet und anschließend den Glycin-Rest mit einer Aminoacylprolin-Aminopep tidase (Aminopeptidase-P,E.C.3.4.11.9.) und den Prolin-Rest mit einer Prolinaminopeptidase (E.0.3.4.11.5.) entfernt.2) Process for the production of proteins according to claim 1, characterized characterized in that the Xyz-Gly bond in the tetrapertide sequence with a Collagenase (E.C.3.4.24.3.) Selectively cleaves and then the glycine residue with it an aminoacylproline aminopeptidease (aminopeptidase-P, E.C.3.4.11.9.) and the proline residue removed with a proline aminopeptidase (E.0.3.4.11.5.). 3) Verfahren zur Herstellung von Proteinen nach Anspruch 2, dadurch gekennzeichnet, daß man die Abspaltung des Glycin-Restes mit Aminopeptidase-P(E.O.3.4.ll..) und da Prolin-Restes mxt Prolinaminopeptidase (E.C.3.4.11.5.) in einem Schritt vornimmt.3) Process for the production of proteins according to claim 2, characterized characterized in that the cleavage of the glycine residue with aminopeptidase-P (E.O.3.4.ll ..) and since proline residue mxt proline aminopeptidase (E.C.3.4.11.5.) makes in one step.
DE19792922496 1979-05-31 1979-05-31 Protein cpds. e.g. hormones isolation from fusion proteins - by selective enzymatic hydrolysis of tetra:peptide sequences contg. proline and glycine Withdrawn DE2922496A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DE19792922496 DE2922496A1 (en) 1979-05-31 1979-05-31 Protein cpds. e.g. hormones isolation from fusion proteins - by selective enzymatic hydrolysis of tetra:peptide sequences contg. proline and glycine
IL60184A IL60184A (en) 1979-05-31 1980-05-29 Process for the specific cleavage of protein sequences from proteins
JP7158880A JPS5668399A (en) 1979-05-31 1980-05-30 Production of protein
EP80730038A EP0020290B1 (en) 1979-05-31 1980-05-30 Process for the specific splitting-off of protein sequences from proteins
DE8080730038T DE3068508D1 (en) 1979-05-31 1980-05-30 Process for the specific splitting-off of protein sequences from proteins
AT80730038T ATE8411T1 (en) 1979-05-31 1980-05-30 METHODS FOR THE SPECIFIC CLEAVAGE OF PROTEIN SEQUENCES FROM PROTEINS.
US06/395,433 US4543329A (en) 1979-05-31 1982-07-06 Process for the specific cleavage of protein sequences from proteins

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DE19792922496 DE2922496A1 (en) 1979-05-31 1979-05-31 Protein cpds. e.g. hormones isolation from fusion proteins - by selective enzymatic hydrolysis of tetra:peptide sequences contg. proline and glycine

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WO1986007380A1 (en) * 1985-06-04 1986-12-18 Takeda Chemical Industries, Ltd. Process for preparing polypeptide

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JPS5668399A (en) 1981-06-09

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