DE4424171A1 - Use of recombinant Erythrina caffra inhibitor for the purification of serine proteases - Google Patents

Abstract

In a process for purifying serine proteases from a protein mixture the serine protease is bonded to an immobilized polypeptide having the activity of a DE-3 inhibitor from Erythrina caffra, the unbonded portions are removed from the protein mixture, the serine protease is separated from the inhibitor, the immobilized inhibitor is separated from the soluble serine protease, and the serine protease is isolated. The process is characterized in that a polypeptide which is the product of a prokaryotic or eukaryotic expression of an exogenic nucleic acid is used as the polypeptide. This inhibitor is noted for its improved specific activity and is suitable in particular for purifying plasminogen activators, such as tissue plasminogen activators (t-PA and derivatives).

Description

The invention relates to an improved method for purifying Serinpro tease using recombinant inhibitor DE-3 from Erythrina caffra. Immobilized trypsin inhibitors from Erythrina (ETI) are effective Reagents for the affinity chromatography purification of serine proteases, especially of plasminogen activators (C. Heussen, J. Biol. Chem. 259 (1984) 11635-11638), β-trypsin, α-chymotrypsin and thrombin (S. Onesti et al., J. Mol. Recogn. 5 (1992) 105-114). These trypsin inhibitors have been around for a long time according to known (C. Heussen, Haemostasis 11 (1982) P47 (supplement); F.J. Joubert, Phytochemistry 21 (1982) 1213-1217; F.J. Joubert, Int. J. Biochem. 14 (1982) 187-193).

The inhibitor DE-3 from E. caffra is particularly preferred for the purification of Plasminogen activators are suitable (F.J. Joubert in Thrombosis and Haemostasis 57 (1987) 356-360). In this publication is also the complete amino acid sequence of this inhibitor. DE-3 can be derived from the seed of E. caffra isolated and purified (F.J. Joubert, Int. J. Biochem. 14 (1982) 187-193).

Teixeira et al., Biochimica et Biophysica Acta 1217 (1994) 16-22 disclose a recombinant ETI described, the specific inhibitory activity for Tissue plasminogen activator is 1.7 × 10⁹ U / mmol. The specific In contrast, inhibitory activity of natural ETI is included 1.94 × 10⁹ U / mmol. The same applies to the inhibitory activity towards Trypsin (2.63 x 1012 / 3.21 x 1012). So that is the specific inhibitory Activity of Teixeira-made recombinant ETI for trypsin and Tissue plasminogen activator is 20% less than the activity of natural ETI.  

Recombinant ETI is obtained by expression and according to Teixeira purified by ammonium sulfate precipitation (80% saturation), dialysis against Water and a cyanogen bromide cleavage, showing the N-terminal sequence including the methionine is split off. Then one Affinity chromatography column (Sephadex G50) chromatographed.

The object of the invention is to improve the effectiveness of methods for Purification of serine proteases using ETI.

The invention relates to a process for the purification of serine proteases from a protein mixture by binding the serine protease to the immobilized ten inhibitor DE-3 from Erythrina caffra, removing the unbound portions the protein mixture, detachment of the serine protease from the inhibitor, separation of the immobilized inhibitor of the soluble serine protease and isolation of the Serine protease, which is characterized in that a DE-3 inhibitor Inhibitor is used which is the product of a prokaryotic or is eukaryotic expression of an exogenous DNA and chromatographic via an anion exchanger, cation exchanger or a nickel chelate acid is cleaned.

Surprisingly, it was found that recombinant, according to the invention manufactured inhibitor DE-3 from E. caffra in contrast to that from natural Sources isolated inhibitor of a significantly increased specific affinity for Possesses serine proteases.

The method according to the invention is particularly advantageous for cleaning Plasminogen activators such as tissue plasminogen activators (t-PA) and Deri vate (e.g. mutations and deletions) thereof. t-PA and derivatives are for example in EP-B 0 093 619, USP 5,223,256, WO 90/09437 and T.J.R. Harris, Protein Engineering 1 (1987) 449-458.

The preparation of the recombinant inhibitor can according to the expert common methods.  

For this purpose, a DNA sequence is first produced, which is able to To produce protein that has the activity of the inhibitor DE-3. The DNA sequence is cloned into a vector that transfers to a host cell can be replicated there. Such a vector contains in addition to Inhibitor sequence operator elements used to express the DNA sequence required are. This vector, the inhibitor DNA sequence and the Opera contains tor elements is transferred into a vector that is capable of To express DNA of the inhibitor. The host cell becomes cultivated under conditions tiviert, which allow the expression of the inhibitor. The inhibitor turns off won these cells. Suitable measures are taken to ensure that the activator can assume an active tertiary structure in which it Shows inhibitor properties.

It is not necessary for the inhibitor to have the exact amino acid sequence according to SEQ ID NO: 2. Inhibitors which are also suitable in the have essentially the same sequence and polypeptides with activity (Ability to bind to serine proteases, especially to t-PA) of an inhibitor DE-3 from Erythrina caffra. However, the amino acid sequence SEQ ID NO: 2 is preferably used, being in the case of expression in prokaryotic hosts cells, but not after eukaryotic expression, an N-terminal methio nin is included.

Another object of the invention is a nucleic acid sequence which in is essentially identical to nucleotides 9 to 527 of SEQ ID NO: 1 and for a polypeptide with the activity of an inhibitor DE-3 from Erythrina caffra encoded, or a nucleic acid sequence that is involved in the degeneration of the genetic codes encoding the same polypeptide. One is preferred DNA sequence, in particular sequence 9 to 527 of SEQ ID NO: 1. For expression in eukaryotic or prokaryotic host cells contains the nucleic acid sequence at the 5'-end known to the expert eukaryotic or pro caryotic transcription and translation signals.  

The nucleic acid sequence of the inhibitor is preferably identical to that Nucleotides 9 to 527 of SEQ ID NO: 1. However, can help the production of the vectors or optimization of the expression modifications to be appropriate. Such modifications are, for example:

• - Change the nucleic acid to different recognition sequences of restriction enzymes to facilitate the Introduce ligation, cloning and mutagenesis steps
• - Change the nucleic acid to incorporate preferred Codons for the host cell
• - Supplementing the nucleic acid with additional operator elements to optimize expression in the host cell.

The inhibitor is preferably expressed in microorganisms such as E. coli. Expression in eukaryotic cells such as yeast, CHO cells or Insect cells, however, is also possible.

For this purpose, biologically functional plasmids or viral DNA vectors are used that essentially contains nucleotides 9 to 527 of SEQ ID NO: 1 ten, or a nucleic acid sequence which is involved in the degeneration of the encoded genetic codes for the same polypeptide. With such Vectors become stable prokaryotic or eukaryotic host cells transformed or transfected.

The expression vectors must contain a promoter that expresses the expression of the inhibitor protein allowed in the host organism. Such promoters are Known to those skilled in the art and are, for example, lac promoter (Chang et al., Nature 198 (1977) 1056), trp (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), XPL Pro motor (Shimatake et al., Nature 292 (1981) 128) and TS Promotor (U.S. Patent No. 4,689,406). Synthetic promoters such as, for example, are also suitable tac promoter (U.S. Patent No. 4,551,433). Coupled Pros are also suitable motor systems such as T7 RNA polymerase / promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113). Hybrid promoters are also suitable from a bacteriophage promoter and the operator region of the micro  organism (EP-A 0267 851). In addition to the promoter is an effective ribo binding site required. For E. coli this becomes ribosome binding referred to as the Shine-Dalgarno (SD) sequence (Shine et al., Nature (1975) 25434; J. Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, USA).

To improve expression, it is possible to use the inhibitor protein as To express fusion protein. In this case, usually one DNA sequence soft for the N-terminal part of an endogenous bacterial protein or another stable protein encoded at the 5'-end for the inhibitor protein coding sequence fused. Examples of this are lacZ, trpE.

After expression, the fusion proteins are preferably treated with enzymes (e.g. Factor Xa) cleaved (Nagai et al., Nature 309 (1984) 810). More examples of the cleavage sites are the IgA protease cleavage site (WO 91/11520) and Ubiquitin cleavage site (Miller et al., Bio / Technology 7 (1989) 698).

The recombinant protein initially obtained as inactive inclusion bodies can converted into soluble active protein by methods familiar to the person skilled in the art become. For this purpose, the inclusion bodies, for example with guanidine hydro chloride or urea solubilized in the presence of a reducing agent, redu graces the reducing agent z. B. removed by dialysis and preferably under Use of a redox system such as reduced and oxidized glutathione renatured.

Such methods are described, for example, in US Pat. No. 4,933,434, EP-B 0 241 022 and EP-A 0219874.

It is also possible to use the proteins as active proteins from the microorganisms to secrete. For this purpose, a fusion protein is preferably used, which from the signal sequence used for the secretion of proteins in the Host organisms (U.S. Patent No. 4,336,336), and nucleic acid, which codes for the inhibitor protein. The protein is either  in the medium (for gram-positive bacteria) or in the periplasmic Space (in Gram-negative bacteria) secreted. Between the signal sequence and the sequence coding for the inhibitor is expediently a cleavage site attached either during processing or in an additional step allows the inhibitor protein to be split off. Such signal sequences are for example ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437), and phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).

The vectors also contain terminators. Terminators are DNA sequences that signal the end of a transcription process. You draw are usually characterized by two structural characteristics: an inversely repetitive one G / C-rich region that can intramolecularly form a double helix and one Number of U (or T) residues. Examples are trp attenuator and terminator in the DNA of the phage fd and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127).

In addition, the expression vectors usually contain a selectable one Markers to select transformed cells. Such selectable markers are, for example, the resistance genes for ampicillin, chloramphenicol, Erythromycin, kanamycin, neomycin and tetracycline (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). The genes are also suitable selectable markers for essential substances of the biosynthesis of substances necessary for the cell such as B. histidine, tryptophan and leucine.

A variety of suitable bacterial vectors are known. Example Vectors have been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. Acad. Sci. USA 79 (1982) 5582), E. coli (Aman et al., Gene 40 (1985) 183; Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Patent No. 4,747,056).

In addition to prokaryotic microorganisms, expression of the inhibitor protein is also possible in eukaryotes (such as CHO cells, yeast or insect cells). Yeast and insect cells are preferred as the eukaryotic expression system. Expression in yeast can be via three types of yeast vectors (integrating YI _P (yeast integrating plasmids) vectors, replicating YR _P (yeast replicon plasinids) vectors and episomal YE _P (yeast episomal plasmids) vectors. More on this is for example in SM Kingsman et al.) Tibtech 5 (1987) 53-57.

Other genetic engineering processes for the production and expression of geeig Neten vectors are in J. Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, USA).

After manufacturing, recombinant ETI is cleaned chromatographically over an anion exchanger, e.g. B. a Q-Sepharose® Column, a cation exchanger (e.g. based on sulfopropyl) or via a Nickel chelate column such as in Porath, J. & Olin, B., Biochemistry 22 (1983), 1621-1630. Surprisingly, after this Purification method obtained a recombinant ETI, its inhibitory Activity against serine proteases such as trypsin and Tissue plasminogen activator is significantly higher than the inhibitory activity of natural ETI.

A polypeptide thus produced and purified has the properties of a Inhibitors DE-3 from Eryrina caffra and is available by cultivating prokaryotic or eukaryotic host cells with an exogenous DNA sequence which is essentially the sequence of nucleotide 9 to 527 of SEQ ID NO: 1, or a sequence that occurs as part of the degeneration of the genetic code encoded for the same polypeptide in a way transformed or transfected that allows the host cells to appropriate nutrient conditions to express the polypeptide, and isolation of the desired polypeptide, which has a specific inhibitory activity 1.07 U / mg or higher against trypsin. This activity is after chromatographic purification on an anion exchanger, Cation exchanger or obtained on a nickel chelate column.  

Another object of the invention is a method for producing a recombinant ETI by culturing prokaryotic or eukaryotic host cells which have an exogenous DNA sequence which is in the essentially corresponds to the sequence nucleotide 9-527 of SEQ ID NO: 1, or a sequence that is part of the degeneration of the genetic code for the same polypeptide encoded, transformed or transfected in a way that it allows the host cells to do this under appropriate nutrient conditions Expression of the polypeptide, isolation of the polypeptide from the host cells and chromatographic purification on an anion exchanger, cation exchanger or on a nickel chelate column.

Purification of serine proteases using recombinant ETI is carried out according to the methods familiar to the person skilled in the art (cf. e.g. F.J. Joubert (1987)). For this, ETI is covalently attached to a matrix (e.g. CNBr-Sepharose column) bound and the protein mixture, which contains the serine protease, at neutra len or weakly alkaline conditions on the column and a Chromatography performed. Elution takes place by lowering the pH  pH 5.5 or by using buffer solutions which are chaotropic agents zien, such as B. KSCN included. The eluate has a protein purity of over 95% based on the serine protease. For further use the serine protease Conveniently transferred to the desired buffer solution by dialysis.

The immobilization of the inhibitor and all further process steps for the Rei Purification of serine protease and t-PA can be carried out in a manner analogous to that for the E. caffra isolated inhibitor DE-3 can be performed. Such procedures are described for example in EP-B 0218 479, EP-B 0 112 122, USP 4,902,623. The immobilization is expediently carried out on an inert carrier ger, preferably on CNBr-Sepharose®.

The following examples and the sequence protocols describe the Erfin extension.  

example 1 Expression of ETI in E. coli a) Gene synthesis

Using the codons preferred by E. coli, the amino acid sequence of the ETI from Erythrina caffra (Joubert and Dowdle, Thrombosis and Haemostasis 57 (3) (1987) 356-360) a corresponding nucleic acid sequence derived and according to the method of Beattie and Fowler (Nature 352 (1991) 548-549) synthetically represented. To facilitate cloning, the 5'-end an interface for the restriction enzyme EcoRI and at the 3'-end one Interface for the restriction enzyme HindIII inserted. The synthesized Nucleic acid was restricted with the enzymes EcoRI and HindIII and with the cloning vector pBS + (Stratagene, US, Catalog No. 211201, derivative the phage f1 and Stratagene's pBS plasmid with T₃ and T₇ promoter gene, Ampicillin resistance gene, f1 origin, ColE-1 origin, lacI gene, lacZ gene and one Multiple cloning site), previously digested with EcoRI and HindIII, ligated. The ligation mixture was transformed into Escherichia coli. Get the Clones selected for ampicillin were restricted by restriction Enzymes EcoRI and HindIII analyzed. The resulting clone, pBS + ETI, ent maintains an additional 539 bp EcoRI / HindIII fragment SEQ ID NO: 1.

b) expression vector

The plasmid pBS + ETI was with the restriction enzymes EcoRI and HindIII restricted, and the 539 bp fragment was isolated. The expressions vector pBTac1 (from Boehringer Mannheim GmbH, catalog No. 1081365) Basis from pUC8, H. Haymerle et al., Nucl. Acid Res. 14 (1986) 8615-8624) was also digested with the enzymes EcoRI and HindIII and that 4.6 kb vector fragment isolated. Both fragments were ligated and together with the helper plasmid pUBS520 (Brinkmann et al., Gene 85 (1989) 109-114), which contains the lac repressor gene, in E. coli (DSM 5443)  transformed. The clones were selected by means of the plasmids mediated ampicillin or kanamycin resistance. The plasmid obtained pBTETI contains an additional one compared to the starting vector pBTac1 EcoRI / HindIII fragment with a size of 539 bp.

Analogously, instead of DSM 5443, DSM 3689, which is already a Contains plasmid can be used. In this case, the helper plasmid pUB520 not required.

c) Expression of recombinant ETI (recETI) in E. coli

The E. coli strain DSM 5443 was used to check the expression performance with the plasmids pBTETI and pUBS520 in LB medium (Sambrook et al., Molecular Cloning (1989) Cold Spring Harbor) in the presence of ampicillin and Kanamycin (50 µg / ml final concentration each) to an optical density (OD) of 0.6 at 550 µm. Expression was determined by adding 5 mM IPTG initiated. The culture was incubated for an additional 4 hours. in the The E. coli were then collected by centrifugation and in buffer resuspended (50mM Tris-HCl pH 8, 50mM EDTA); by sound brought about the lysis of E. coli. By centrifugation again Insoluble protein fractions (inclusion bodies) collected and in the above resuspended by sonication. The suspension was 1/4 Volume of application buffer (250 mM Tris-HCl pH 6.8, 0.01 M EDTA, 5% SDS, 5% mercaptoethanol, 50% glycerin and 0.005% bromophenol blue) are added and analyzed using a 12.5% SDS polyacrylamide gel. As a control, the same preparation with a culture of E. coli (pBTETI / pUBS520) that was not IPTG had been added, carried out and applied to the polyacrylamide gel In the preparation of the IPTG-induced culture one can, after staining of the gel with 0.2% Coomassie-Blue R250 (dissolved in 30% methanol and 10% Acetic acid) and decolorization of the gel in a methanol-acetic acid mixture, a recognize clear band with a molecular weight of about 22 kD. These There is no band in the preparation of the uninduced E. coli cells to find.  

Example 2 Renaturation and cleaning of recETI

50 g of inclusion bodies (IBs) were solubilized with 0.1 M Tris / HCl, pH 8.5, 6 M guanidine, 0.1 M DTE, 1 mM EDTA (90 min at 25 ° C., C _Prot. = 10 mg / ml) and dialyzed after adjusting the pH to 2.5 (HCl) against 3 mol / l guanidine / HCl. The dialysate was centrifuged (SS34, 13,000 rpm) and adjusted to C _Prot. = 36.9 mg / ml by concentration over YM 10. A 1 liter reaction vessel was filled with 0.1 M Tris / HCl, pH 8.5, 1 mM EDTA, 1 mM GSH, 0.1 mM GSSG. The renaturation was carried out at 20 ° C. by adding the dialysate 16 times (600 μg protein / ml buffer each) at intervals of 30 min.

The renaturation results in 2.8 U / ml active ETI.

Cleaning of recETI a) Via anion exchangers

recETI is in 0.1 M Tris / HCl, pH 8.5, 1 mM EDTA, 1 mM GSH, 0.1 mM GSSG renatured. The renaturate is diluted 1: 2 with H₂O, to pH 8.0 with HCl adjusted, dialyzed against 50 mM Tris / HCl pH 8.0 and to a 50 mM Tris / HCl, pH 8.0 equilibrated Q-Sepharose® column applied (5 mg protein / ml Gel). After washing the column with the equilibration buffer and with 50 mM Na₂HPO₄ / H₃PO₄, pH 8.0 (five column volumes each) is carried out with the elution 50 mM Na₂HPO₄ / H₃PO₄, pH 8.0, 0.2 M NaCl.

b) Via cation exchangers

Renatured ETI was adjusted to pH 4.0 by adding HCl and counter 50 mM NaOAc / HCl, pH 4.0 dialyzed (cross flow). The dialysate was centrifuged (13,000 rpm, 30 min, SS 34) and on a with 50 mM NaOAc / HCl, pH 4.0 equilibrated TSK-SP column (cation exchanger with  Sulfopropylseitengruppen, Merck, Germany, volume 15 ml) applied. After washing the column with the equilibration buffer and with 50 mM NaOAc / HCl, pH 4.0, 0.1 M NaCl, the elution is 50 mM NaOAc / HCl, pH 4.0, 0.2 M NaCl.

The purity of the eluate was checked by SDS-PAGE and by RP-HPLC.

Result

Under the conditions used, ETI binds to the TSK-SP column and can be eluted with 0.2 M NaCl. SDS-PAGE and RP-HPLC analysis result a purity of) 95%.

Example 3 Comparison of the specific activity of recETI and ETI from the semen by Erythrina caffra

recETI and ETI, isolated from the seed of E. caffra, were compared to 50 mM Na₂HPO₄ / H₃PO₄4, pH 8.0, 0.2 M NaCl and dialyzed Protein concentration set at 0.8 mg / ml. The protein concentration was determined by measuring the UV absorption at 280 nm (ε = 1.46 cm² / mg).

Determination of ETI activity

The inhibition of trypsin by ETI is with N-α-Benzoylethylester (BAEE) measured as a substrate. 40 ul of a trypsin solution (0.13 mg / ml 2 mM HCl) who that in a quartz cuvette with 60 µl test buffer (0.1 M Tris / HCl, pH 8.0) and Mix 100 µl ETI solution and incubate for 5 min at 30 ° C. After adding The absorbent becomes 800 µl BAEE solution (20 mg BAEE × HCl / 100 ml test buffer) tion increase / min determined at 253 nm.  

The ETI activity is determined using the following formula:

U / ml = [1-E _sample / E _trypsin ] · C _trypsin · 0.328 · V

E _sample : increase in extinction / min of the inhibited sample
E _Trypsin : increase in absorbance / min of uninhibited trypsin
C _Trypsin : Trypsin concentration in the test mixture
V: predilution of the ETI solution

Result

The specific activity of recETI is 20% higher than the specific one activity of the E. caffra iso iso ETI.

Example 4 Coupling recETI to CNBr-Sepharose®

170 mg of purified recETI were against 0.05 M H₃BO₃ / NaOH, pH 8.0, 0.5 M NaCl (coupling buffer) dialyzed and with 7.5 g CNBr-Sepharose® (overnight swollen in 500 ml 1 mM HCl, then aspirated and sus in Susp commutes) mixed. The suspension was incubated for 90 min at room temperature, suction filtered and shaken overnight with 400 ml 0.1 M Tris / HCl, pH 8.0. The recETI-Sepharose® was suctioned off and with 0.7 M arginine / H₃PO₄, pH 7.5 equilibrated.  

Example 5 Purification of a recombinant plasminogen activator

54 mg of recombinant plasminogen activator K2P (produced according to WO 90/09437 or USP 5,223,256) were equilibrated with 0.7M arginine / H₃PO₄, pH 7.5 given recETI-Sepharose. After washing with the equilibration buffer and with 0.3 M arginine / H₃PO₄, pH 7.0 (five column volumes each) Elution with 0.3 M arginine / H₃PO₄, pH 4.5. The plasminogen activator content in Eluate was determined with S 2288 as substrate (Kohnert et al., Prot. Engineer. 5 (1992) 93-100).

Result

The binding capacity of the recETI-Sepharose for the plasminogen acti vator is 1.2 mg (corresponding to 0.63 MU) plasminogen activator / ml recETI-Sepharose.

Claims (19)

1. Process for the purification of serine proteases from a protein mixture by binding the serine protease to the immobilized inhibitor DE-3 from Erythrina caffra, removing the unbound portions from the protein mixture, detaching the serine protease from the inhibitor, separating the immobilized inhibitor from the soluble serine protease and Isolation of the serine protease, characterized in that an inhibitor is used as the inhibitor DE-3, which is the product of a prokaryotic or eukaryotic expression of an exogenous DNA and is purified chromatographically via an anion exchanger, cation exchanger or via nickel chelate. 2. The method according to claim 1, characterized in that the serine protease is a plasminogen activator. 3. The method according to claim 2, characterized in that the plasminogen activator is a tissue plasminogen activator or a derivative thereof. 4. The method according to claim 1 to 3, characterized in that the inhibi is immobilized on an anion exchanger. 5. The method according to claims 1 to 4, characterized in that the exogenous DNA sequence essentially of the sequence nucleotide 9 to 527 from SEQ ID NO: 1 or a sequence which is part of the degeneration of the encodes genetic codes for the same polypeptide. 6. Use of an inhibitor DE-3 from Erythrina caffra, which the product a prokaryotic or eukaryotic expression of an exogenous DNA and is chromatographic via an anion exchanger, Cation exchanger or cleaned over nickel chelate chromatographic purification of serine proteases.   7. Use according to claim 6, characterized in that the serine protease is a plasminogen activator. 8. Use according to claim 7, characterized in that the plasmino gene activator is a tissue plasminogen activator or a derivative thereof. 9. Process for the preparation of a polypeptide which has the properties of an inhibitor DE-3 from Erythrina caffra, by cultivating prokaryotic or eukaryotic host cells with an exogenous DNA sequence which is essentially the sequence of nucleotide 9 to 527 from SEQ ID NO: 1 or a sequence which is part of the degeneration of the encodes genetic codes for the same polypeptide, corresponds to one Are transformed or transfected in ways that allow host cells to to express the polypeptide under appropriate nutrient conditions, and Isolation of the desired polypeptide, which is characterized is that it has a specific inhibitory activity of about 1.07 U / mg against Trypsin owns. 10. The method according to claim 9, characterized in that as DNA sequence, the sequence nucleotide 9 to 527 from SEQ ID NO: 1 or one Sequence that is part of the degeneration of the genetic code for the encoded same polypeptide is used. 11. The method according to claim 9 or 10, characterized in that the Host cells are E. coli cells. 12. The method according to claim 9 or 10, characterized in that the Host cells are yeast cells or CHO cells. 13. DNA sequence of nucleotides 9 to 527 from SEQ ID NO: 1, which for a Polypeptide with the activity of an inhibitor DE-3 from Erythrina caffra coded.   14. Biologically functional plasmid or viral DNA vector which is a Contains DNA sequence according to claim 13. 15. Prokaryotic or eukaryotic host cells, which with a DNA vector according to claim 14 are stably transformed or transfected. 16. Polypeptide which has the properties of an inhibitor DE-3 from Erythrina caffra owns and is obtainable by cultivating prokaryotic or eukaryotic host cells that have an exogenous DNA sequence, which essentially the sequence nucleotide 9 to 527 from SEQ ID NO: 1 or a sequence that is part of the degeneration of the genetic code for the same polypeptide encodes, conforms, transforms in a manner or are transfected, which allows the host cells to take appropriate nutrient conditions to express the polypeptide, and isolation of the desired ten polypeptide, which is characterized in that it has a specifi has inhibitory activity of approx. 1.07 U / mg against trypsin. 17. A polypeptide according to claim 16, characterized in that the expression in prokaryotic host cells and the polypeptide the amino has acid sequence SEQ ID NO: 2. 18. Polypeptide according to claim 16, characterized in that the expression in eukaryotic host cells and the polypeptide the amino acid sequence SEQ ID NO: 2 without N-terminal methionine. 19. A method for producing a recombinant ETI by cultivating prokaryotic or eukaryotic host cells with an exogenous DNA sequence which is essentially the sequence of nucleotide 9 to 527 of SEQ ID NO: 1, or a sequence that occurs as part of the degeneration of the genetic code encoded for the same polypeptide in a way transformed or transfected that allows the host cells to appropriate nutrient conditions to express the polypeptide, isolation of the polypeptide from the host cells and chromatographic purification an anion exchanger, cation exchanger or on one Nickel chelate column.