DE19627162C1 - New covalently crosslinked imprint poly:peptide, e.g. enzyme - Google Patents

Abstract

The invention relates to imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and uses thereof. The properties of said imprint polypeptides (VIP) covalently cross-linked are present in aqueous and in organic solvent systems, and can therefore be used in both types of system.

Description

Field of application of the invention

The invention relates in its conformation to fixed and stabilized, covalently cross-linked Imprint polypeptides (embossed polypeptides), e.g. B. Proteins used in catalytic synthesis, Chromatography and analysis of chiral compounds as well as in biosensor technology for specific recognition of molecules can be used, methods for their Manufacture and its use.

State of the art

Polymers with the property for the selective recognition of molecules are in the Biotechnology and chemistry of the greatest industrial importance. These include the Substance class of protein-counting enzymes, antibodies and receptors. These polymers possess due to their three-dimensional molecular structure (conformation) for certain Molecules (substrates, antigens, hormones etc.) complementary areas that as Binding sites are called. Because native polymers are often commercially available bind interesting molecules inadequately or not at all, or those necessary for binding Conformation is not sufficiently stable under the conditions of use are methods  has been developed to produce tailor-made polymers to the desired Show complementarity (K. Mosbach and O. Ramström, Biotechnology, Vol. 14 (1996), 163-170).

Goldstein, Methods Enzymol. 19 (1970), 935-962, and Fritz et al., Angew. Chem. 78, 1966, 775 describe a method of immobilizing proteins using cyclic anhydrides for covalent coupling. The procedure is first one To copolymerize anhydride to a carrier matrix with other monomers and then couple the protein to the finished carrier.

Mosbach and Ramström (loc. Cit) describe that the production of customized polymers with the desired complementarity is carried out in two variants. Both variants are based on the principle that a specially selected molecule, hereinafter referred to as the print molecule (embossing molecule), acts as a shaping agent (embosser) and is responsible for the complementarity desired and thus new property in the polymer. Fig. 1 and 2 schematically illustrate these two variants.

Variant 1 for the production of customized (embossed) polymers is carried out in such a way that a copolymerization of selected monomers is carried out in an organic solvent in the presence of the print molecule (see FIG. 1). First, the print molecule a) is dissolved directly in an organic solvent system together with a monomer (non-covalent method) or b) after derivatization with a monomer (covalent method), then a crosslinker and a radical initiator are added and the polymerization is started. The print molecule must then be extracted from the resulting polymer under suitable conditions so that the new binding sites generated in the polymer are released. The polymer produced in this way has selective recognition sites for the print molecule or for structurally similar molecules (see FIG. 1).

Essential for a) and b) is that both the solution of the starting substances resulting from certain concentrations of print molecule, monomer (s) and radical initiator composed, as well as the tailor-made (embossed) subsequently produced in the polymer Property, previously only on the use of organic solvent systems is instructed.

In variant 2, to produce a customized (embossed) polymer, no polymerization is carried out in the presence of a print molecule, but an already finished native polymer, e.g. B. a polypeptide or a protein, is forced under certain conditions and by the presence of the print molecule to a conformational change (see Fig. 2). For this purpose, the polymer is first dissolved together with the print molecule in an aqueous solvent system and then by adding additives, for. B. organic solvents precipitated. The print molecule and the polymer interact with one another via non-covalent interactions in such a way that the conformation of the polymer is changed and the polymer thereby has a new property which is inherently predetermined by the print molecule. The process of variant 2 described up to this point is hereinafter referred to as imprinting (embossing), the resulting polymer as imprint polymer (embossed polymer); e.g. B. Imprint polypeptide.

However, the new property of the imprint polymer is reversible. It only remains as long as it is used under conditions that the regression to the original prevents thermodynamically favorable conformation. Therefore, through variant 2 obtained polymers, which by Mosbach et al. loc. cit., Ståhl et al., Biotechnol. Lat. 12 (1990), 161-166; Ståhl et al., J.Am. Chem. Soc. 113: 9366-9368 (1991) and Klibanov et al., J.Biol.Chem. 263 (1988), 11624-11626 and Dabulis and Klibanow, Biotechnol.Bioeng. 39 (1992), 176-185 were only used in organic solvents.

There are two options for imprinting (see Fig. 2). One creates reversible binding sites for the print molecule that did not previously exist on the polymer, the second modifies the stereo and / or substrate selectivity of a polymer, e.g. B. an enzyme by the interaction of the print molecule with the active center. The first possibility resulted in the use of bovine serum albumin as a polypeptide and L-malic acid as a print molecule, an imprint polypeptide, which was selective in chromatographic studies for the print molecule L-malic acid (Dabulis and Klibanow, loc. Cit). The second possibility led to extended, new catalytic properties of these enzymes in hydrolases. So z. B. the substrate selectivity of subtilisin (Russel and Klibanow, loc. Cit.) And the stereo and substrate selectivity of α-chymotrypsin (Ståhl et al., Loc. Cit.) The polypeptide α-chymotrypsin, for example, has the property of hydrolyzing the (L) -enantiomer of the N-acetyl-tryptophanethyl ester (N-Ac-TrpEE) in an aqueous solvent system or in an organic solvent system made of N-acetyl-L-tryptophan ( N-Ac-Trp) and ethanol. The (D) enantiomer of N-Ac-TrpEE can neither be hydrolyzed nor synthesized.

However, this polypeptide α-chymotrypsin is imprinted with the print molecule N-Ac- (D) -Trp (coined), it can then synthesize N-Ac- (D) -TrpEE in organic solvent catalyze from N-Ac- (D) -Trp and ethanol because of the conformation of the polypeptide was specifically changed by the print molecule (Ståhl et al., loc. cit.). The However, the change in conformation is reversible. Therefore, the imprint polypeptide cannot aqueous solvent system for the hydrolysis of the N-Ac- (D) -TrpEE be used. The new property is lost when the imprint polypeptide with aqueous Solvent systems comes into contact.

One limitation of the imprinting method used so far is that the Imprint polypeptides are labile and their imprinted only in the organic solvent system (embossed) new properties or keep. The conformation of the Imprint polypeptides are based on sensitive, non-covalent bonds. Once this with watery Solvent systems come into contact are those through imprinting induced properties no longer detectable and irreversibly lost since the induced Conformational change in aqueous systems is energetically unfavorable. Would be covalent They could fix and stabilize the conformation of the imprint polypeptides subsequently also used in an aqueous environment. The imprinted conformation would be fixed covalently and not lost without breaking the knotted bonds. Water molecules could no longer cancel the imprinted conformation. Completely new ones would arise  Perspectives on the use of imprint polypeptides, as a large number of commercial or industrially relevant processes in aqueous solvent systems. For example are many interesting compounds only or better soluble in aqueous systems and the Catalytic activity of polypeptide catalysts is also higher in aqueous systems and can only be used commercially.

For example, B. running in an aqueous environment, the process due to too low or no activity of the available polypeptide catalysts (enzymes) against one commercially usable substrate have not been used so far Fixing the conformation of the imprint polypeptide to a substantial one Lead productivity increase (expansion of catalytic property). Existing procedures with native polypeptides could be fixed in their conformation by using and stabilized imprint polypeptides experience an enormous increase in efficiency. Conformationally fixed imprint polypeptides that selectively the print molecule and / or its structure-like compounds in aqueous systems could be recognized as artificial Antibodies in aqueous analysis, sensor technology or chromatography, such as the Affinity chromatography, specifically used in immunoaffinity chromatography. According to the current state of the art, these applications are mentioned in aqueous systems so far not feasible.

Object of the invention

The invention is therefore based on the object of being fixed in its conformation to provide stabilized, covalently cross-linked imprint polypeptides that are also available in aqueous media can be used. The new or expanded catalysis and / or Binding properties of the imprint polypeptides should be fixed and stabilized. So that is the loss, d. H. the reversibility of the imprinted property (s) of the polypeptides in aqueous media no longer exist. The imprint polypeptides are then also in for the first time catalytic, chromatographic and / or analytical processes used in aqueous media are carried out.

To solve this task, their conformation becomes fixed and stabilized, covalent cross-linked imprint polypeptides, both in aqueous and in organic Solvent systems are stable, provided by one embodiment the following steps are available:

• (A) covalent introduction of polymerizable, unsaturated bonds containing groups into a polypeptide;
• (B) Imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium;
• (C) Precipitation of the polypeptide / print molecule mixture obtained in step (B) by adding an additive suitable for precipitation and / or Freeze-drying the polypeptide / print molecule Ge obtained in step (B) mixed; and  
• (D) copolymerization of the imprint polypeptide obtained in step (C) in one organic solvents with a crosslinker that is compatible with the polymerizable groups containing unsaturated bonds is copolymerizable.

According to another embodiment, the and are fixed in their conformation stabilized, covalently cross-linked imprint polypeptides, those in both aqueous and organic Solvent systems are stable, available through the following steps:

• (A) covalent introduction of polymerizable, unsaturated bonds containing groups in a polypeptide in an aqueous medium;
• (B) Imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium; and
• (C) copolymerization of the polypeptide obtained in step (B) in an aqueous Medium with a crosslinker that is compatible with the polymerizable, unsaturated bonds containing groups is copolymerizable.

Other solutions to the problem are step (B) before step (A) or Perform steps (A) and (B) simultaneously.

Description of the invention

The method according to the invention (see FIGS. 3 and 4) comprises the selective introduction of polymerizable groups containing unsaturated bonds into an optionally imprinted (embossed) polypeptide, e.g. B. a protein. After the desired polypeptide conformation has been set by the imprinting and the precipitation or freeze-drying (step (C)), the process according to the invention comprises the further step (D) that selectively on the groups of the derivatized polypeptides containing unsaturated bonds a covalent crosslinking by copolymerization with a crosslinker, e.g. B. a divinylated compound takes place, which fixes and stabilizes the desired polypeptide conformation covalently. The covalently cross-linked imprint polypeptide is then stable in both aqueous and organic solvent systems and the imprinted property, e.g. B. a changed catalysis and / or affinity property can be used for commercial applications (see Fig. 3 and 4).

Polypeptides with, for example, alkyl-, aryl-, OH-, NH₂-, SH-, and / or COOH groups, on the one hand, with the print molecule via non-covalent Bindings, e.g. B. ion, hydrogen bonds, hydrophobic interactions, van of the Waals forces, metal chelate complexes, interact and secondly before, during or after imprinting can be covalently derivatized with groups that are polymerizable, contain unsaturated bonds.

In a preferred embodiment, polypeptides with OH, NH₂ and / or SH groups used as starting compounds.  

In a further preferred embodiment, native proteins are used as polypeptides used, preferably enzymes, in particular β-glucosidase or α-chymotrypsin.

Preferred enzymes are selected from oxido reductases, such as D- and L-amino acid oxidases, alcohol dehydrogenase, glucose oxidase and formate dehydrogenase. Further preferred enzymes are selected from hydrolases, such as proteases, peptidases (e.g. Rennin), Amylases (e.g. α-amylase, β-amylase, glucoamylase, β-galactosidase), glycosidases, Acylases, lipases and esterases.

Furthermore, preferred enzymes can be selected from isomerases such as Glucose isomerase and amino acid racemas.

Polymerases, such as DNA polymerases, transferases, such as phosphotransferases or ligases such as DNA ligases can be used.

The introduction of the polymerizable groups containing unsaturated bonds is described in carried out in an aqueous medium. The pH of the aqueous medium is preferably about 3 to 12, preferably about 5 to 8 and in particular about 6 to 8. If appropriate the aqueous medium can contain a buffer system. The buffer system is preferred selected from a potassium phosphate buffer, a citric acid phosphate buffer, Tris buffer (tris (hydroxymethyl) aminomethane) or a sodium phosphate buffer.

The process according to the invention is preferably characterized in that the introduction of the polymerizable groups containing unsaturated bonds, preferably vinyl groups, has to be carried out in a polypeptide in a selective manner. Part of the functional groups of the polypeptides (OH, NH₂, SH, COOH groups), which is to interact with a print molecule during the imprinting in the aqueous solvent system, should not be derivatized. This can be controlled, for example, by the molar ratio of polypeptide / olefinic compound. The degree of derivatization is determined by the respective primary sequence of the polypeptide, the amino acid composition, the temperature and the pH. The optimal degree of derivatization for maintaining the imprinted property can easily be checked by comparing the underivatized imprint polypeptide with the differently derivatized imprint polypeptides with regard to the desired property (s) ( FIG. 5).

Preferred mole ratios of polypeptide / olefinic compound are in the range of about 1: 5 to 1: 300.

In a preferred embodiment of the process according to the invention, olefinic ones are used Compound or mixtures thereof, selected from the group consisting of reactive Analogs or derivatives of the general formula (I)

used, the radicals R₁, R₂, R₃ and R₄ independently of one another hydrogen atoms, Carboxyl residues, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl radicals, alkoxy radicals or carboxyalkyl radicals with preferably up to 10, more preferably up to 6, in particular 1 or 2 carbon atoms or substituted or are unsubstituted aryl radicals or carboxyaryl radicals, with the proviso that at least one the radicals R₁, R₂, R₃ and R₄ independently of one another a carboxyl, carboxyalkyl or Is carboxyaryl.

Preferably 1 or 2, in particular 2 of the radicals R₁, R₂, R₃ and R₄ are independent a carboxyl, carboxyalkyl or carboxyaryl radical, in particular carboxyl or Carboxyalkyl radicals with in particular 1 or 2 carbon atoms.

Preferably, the radicals R₁, R₂, R₃ or R₄, which are not carboxyl, carboxyalkyl or Carboxyaryl, independently of one another, are more preferred hydrogen atoms or alkyl radicals Ethyl or methyl radicals, especially methyl radicals.

If the radicals R₁, R₂, R₃ and R₄ substituted alkyl, carboxyalkyl, alkoxy, aryl or Are carboxyaryl radicals, the substituents are preferably selected from the group consisting of halogen atoms, nitro, amido, carboxyl, ester and alkoxy radicals with preferably up to 10, more preferably up to 5 and in particular 1 or 2 carbon atoms.

If the radicals R₁, R₂, R₃ and R₄ are unsaturated alkyl, carboxyalkyl or alkoxy radicals, they preferably contain up to 5, more preferably up to 3 and in particular an unsaturated one Binding.

If the radicals R₁, R₂, R₃ and R₄ substituted or unsubstituted aryl radicals or Are carboxyaryl radicals, the aryl radicals are preferably phenyl or naphthyl radicals, the Substituents are preferably selected from those for substituted alkyl, Group called carboxyalkyl, alkoxy, aryl or carboxyaryl radicals.

In a preferred embodiment, compounds are selected from the group consisting of itacon, maleic, citracon, croton, methacrylic and acrylic acid used.

Preferably olefinic cyclic or non-cyclic anhydrides, such as itacon, Maleic, citraconic or crotonic, acrylic or methacrylic anhydride, or the Acid halides, especially the acid chlorides of the olefinic compounds mentioned Derivatization used.

In a further preferred embodiment, itaconic anhydride (ISA) is used as the olefinic compound in an aqueous medium, preferably in the pH range from about 3-12, which binds selectively and covalently to OH, NH₂ and / or SH groups (cf. Tab. 3 ). The number of ISA bonds is determined by the individual primary sequence of the polypeptide, the temperature and the pH. The optimal percentage of possible derivatization for maintaining the imprinted property is checked. For this purpose, the non-derivatized imprint polypeptide is compared with the imprint polypeptides derivatized to different degrees in terms of the desired property (s) ( FIG. 5).

In a further preferred embodiment of the method according to the invention Stabilization of the desired conformation or to shield it for imprinting important functionalities during the derivatization process (step (A)) polyols, such as Polyethylene glycols, sorbitol, sucrose, glucose and / or glycerin or salts added, such as Ammonium, alkali or alkaline earth metal sulfates, phosphates or halides, such as (NH₄) ₂SO₄, Na₂SO₄, MgSO₄, Na₃PO₄, CaHPO₄, (NH₄) H₂PO₄, NH₄Cl, NaCl, KCl and CaCl₂. So owns this process step many degrees of freedom in order to be a functionally active and derivatized imprint polypeptide.

The inventive method is also characterized in that the polypeptide before, during or after the covalent introduction of polymerizable, unsaturated Groups containing bonds is incubated with a print molecule in the aqueous medium. The print molecule and the polypeptide form a polypeptide / print molecule mixture, whereby the print molecule and the polypeptide interact via non-covalent interactions. This changes the conformation of the polypeptide, the new and / or has changed properties.

A compound which has a high concentration can preferably be used as the print molecule structural and / or chiral similarity with the later substrate of the finished one in its Conformationally fixed and stabilized, covalently cross-linked imprint polypeptide or with the later substrate is identical.

In a preferred embodiment, α-chymotrypsin is used as the polypeptide and as Print molecule N-Ac- (D) -Tryptophan, N-Ac- (D) -Tyrosin or N-Ac- (D) -Phenylalanin used.

In a further preferred embodiment, bovine serum albumin and L-malic acid is used as the print molecule or D- or L-amino is used as the polypeptide acid oxidase and as a print molecule are natural or non-natural (D) - or (L) amino acids used.

In addition, the inventive method is characterized in that the in one aqueous medium dissolved polypeptide / print molecule mixture is precipitated. The Precipitation can be done by adding additives that are used to precipitate the Polypeptide / print molecule mixture in an aqueous medium are suitable.

In a preferred embodiment, water-miscible organic solvents are used as Additives used for precipitation, preferably acetone, methyl ethyl ketone, methanol, Ethanol, propanol and butanol, especially n-propanol or isopropanol.

In a further embodiment, the polypeptide / print molecule mixture freeze-dried.

In a further preferred embodiment, the polypeptide / print molecule mixture both precipitated and freeze-dried.  

The imprinted group is printed over the polymerizable groups containing unsaturated bonds Conformation of the polypeptide in a further step by covalent copolymerization fixed.

The copolymerization is preferably carried out by UV radiation, peroxides or radical initiators (thermally or by UV radiation) initiated.

Preferred radical initiators are azobis compounds, such as α, α′-azo-isobutvronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) -valeronitrile (ABDV) used. Especially AIBN or ABDV and the copolymerization are preferably used as radical initiators is initiated by means of UV radiation.

The inventive method is further characterized in that for the covalent Fixation of the conformation after precipitation and / or freeze-drying obtained derivatized imprint polypeptides, a crosslinker is used.

In a preferred embodiment of the method according to the invention, Crosslinker compounds containing unsaturated bonds or mixtures thereof, selected from the group consisting of analogs or derivatives of the general formula (II)

H₂C = C (R₁) - (R₂) _n -C (R₃) = CH₂ (II)

used, the radicals R₁ and R₃ independently of one another hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxyl, Carboxyalkyl or alkyl ether radicals with preferably up to 10, in particular 1 or 2 Are carbon atoms or substituted or unsubstituted aryl radicals and the radical R₂ cyclic or linear, saturated or unsaturated, substituted or unsubstituted Alkyl or alkyl ether radicals with preferably up to 10, in particular 1 or 2 Is carbon atoms or an aryl radical and n has a value of preferably up to 10 and especially 1 or 2.

The radicals R 1 and R 3 are preferably hydrogen atoms.

In a further preferred embodiment, the radicals R₁ and / or R₃ can more than 10 Contain carbon atoms. In a further preferred embodiment, n has a value greater than 10. Specific examples are olefinic polyethylene glycols or olefinic ones Derivatives of polyacrylamides.

If the radicals R₁, R₂ and R₃ are unsaturated and / or substituted radicals, they have the the general formula (I) meaning. In addition to the substituents mentioned Hydroxy and / or amino residues can be present.

The crosslinkers used are preferably selected from the group consisting of Divinylalkyl and divinylaryl compounds, especially divinylbenzene or 1,5-hexadiene.  

In a further preferred embodiment, the unsaturated bonds used containing crosslinker or mixtures thereof selected from the group consisting of Analogs or derivatives of the general formula (III)

the radicals R₁, R₂ and R₃ and n being those mentioned above for the general formula (II) Have meaning and the radical X is an oxygen atom or the group -NH.

The crosslinkers are preferably selected from ethylene glycol dimethacrylate, N, N'-methylene-bis-acrylamide, diitaconic acid alkyl amides and diitaconic acid arylamides.

In a preferred embodiment, the copolymerization in cyclohexane, toluene, Acetone, alkanols, ethyl acetate, tetrahydrofuran, chloroform or mixtures thereof, in particular carried out in cyclohexane, preferably ethylene glycol dimethacrylate, Divinylbenzene, diitaconic acid alkylamides, -arylamides, divinylalkyl- or -aryl compounds as Crosslinker can be used.

In a preferred embodiment, the derivatized imprint polypeptide obtained after the precipitation step (see FIGS. 3 and 4) is suspended in an organic solvent, one or more of the above-mentioned crosslinkers are added, the radical initiator, e.g. B. azobis compounds (α, α'-azo-isobutyronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) valeric acid nitrile (ABDV)), added and the copolymerization started by UV radiation. The polypeptide conformation is fixed covalently via the previously introduced olefinic double bonds; the important free functionalities of the imprint polypeptide are not blocked by this step. The imprinted conformation is thus covalently fixed and can be used in the aqueous and organic solvent system.

The imprint polypeptide, which is fixed and stabilized in its conformation, is covalently cross-linked usable under many conditions under which there is no selective introduction of residues and the copolymerization would have been unstable, e.g. B. in aqueous solvent systems.

In a further embodiment, a polypeptide is obtained by the covalent introduction of polymerizable groups containing unsaturated bonds derivatized. The polypeptide can be selected from the above group. The derivatization can be like for step (A) described above.

The polypeptide is then imprinted with a print molecule in an aqueous medium (embossed). The print molecule can be selected as described above. A native enzyme is preferably used as the polypeptide and the substrate as the print molecule Enzyme used.  

The copolymerization is then carried out in an aqueous solvent with a Crosslinker carried out with the polymerizable, containing unsaturated bonds Groups are copolymerizable. The copolymerization is carried out as above for step (D) described. The crosslinkers are preferably from the above described groups selected. The crosslinker is chosen so that it is in the aqueous Solvent is soluble. Preferably, the crosslinker is N, N'-methylene-bisacrylamide used.

The invention is illustrated by the following examples.

example 1 Gentle introduction of olefinic groups into polypeptides A. Vinylation of α-chymotrypsin (EC 3.4.21.1) with itaconic anhydride

Α-Chymotrypsin is dissolved in 10 ml of 0.05 M potassium phosphate buffer pH 7.8 with stirring. Itaconic anhydride is then added in spatula tips at room temperature. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M potassium phosphate buffer pH 7.8 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized. The enzymatic activity of α-chymotrypsin is determined by means of the hydrolysis of benzoyl-L-tyrosine ethyl ester in a photometric test. The extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay) (Habeeb AFSA (1966), Anal. Biochem. 14, 328-336). Tab. 1 shows the reaction batches, the extent of the modification of the functional groups and the enzymatic activities of the α-chymotrypsin derivatives in relation to the free enzyme.

Table 1

Derivatization of α-chymotrypsin with itaconic anhydride

B. Vinylation of the β-glucosidase from the almond (EC 3.2.1.21) with itaconic anhydride

The β-glucosidase is dissolved in 10 ml of 0.05 M citric acid-phosphate buffer pH 6.0 with stirring. D (+) - glucose can be added to stabilize the enzyme. Itaconic anhydride is then added in spatula tips at room temperature. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M citric acid-phosphate buffer pH 6.0 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized. The enzymatic activity of β-glucosidase is determined by means of the hydrolysis of 4-nitrophenyl-β-D-glucopyranoside in a photometric test. The extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay). Table 2 shows the reaction approaches, the extent of the modification of the functional groups and the enzymatic activities of the β-glucosidase derivatives in relation to the free enzyme. To protect the active center of the polypeptide, glucose was added as an additive. In the presence of 3M glucose, 99% residual activity could be obtained after derivatization.

Table 2

Derivatization of β-glucosidase with itaconic anhydride

Example 2 Reactivity of itaconic anhydride with the side chains of polypeptides

Various α-N-acetyl- (L) -amino acids are in 5 ml of 0.05 M citric acid-phosphate buffer pH 7.5 dissolved, mixed with itaconic anhydride while stirring and at 1 hour Incubated at room temperature. The TNBS assay is performed with a sample of each in the buffer dissolved α-N-acetyl- (L) -amino acid and with the incubated with itaconic anhydride α-N-Acetyl- (L) -amino acid performed.  

It can be noted that itaconic anhydride with the amino groups of α-N-acetyl- (L) -lysine and α-N-acetyl- (L) -glutamine, with the sulfhydryl group of α-N-acetyl- (L) -cysteine , can react with the hydroxy groups of α-N-acetyl- (L) -tyrosine and α-N-acetyl- (L) -serine (Tab. 3 ). The reactivities of the itaconic anhydride to the amino acid side chains were Ser <Tyr <Glu <Lys <Cys.

Table 3

Investigations on the reactivity of itaconic anhydrida

On the stability of the above It should be noted that, for example, primary amino groups with itaconic anhydride lead to amide linkages ranging from pH 1-12 and at temperatures are stable up to 70 ° C (R. Kölle, 1995, dissertation, TU Braunschweig, Germany, 53-59).

Example 3 Preparation of a fixed, imprint polypeptide

30 mg of the α-chymotrypsin derivatized with itaconic anhydride from Example 1 (extent of modification = 70%) are dissolved in 1 ml of 0.01 M sodium phosphate buffer pH 7.8. The buffer contains N-Ac- (D) -Trp in a concentration of 0.02 M. The solution is cooled to 0 ° C and mixed with 4 ml of 1-propanol, which has been cooled to -20 ° C. The resulting precipitate is centrifuged, washed with 10 ml of 1-propanol and finally lyophilized. 340 mg of ethylene glycol dimethacrylate and 4 mg of 2,2'-azo-bis- (2-methylpropiononitrile) are dissolved in 0.5 ml of cyclohexane. 5 mg of lyophilisate are suspended in it. The copolymerization is initiated by means of UV light. The copolymerization period is 4 hours. The covalently cross-linked and imprinted α-chymotrypsin (cross-linked, imprint polypeptide; VIP) is washed with cyclohexane and lyophilized (see FIG. 3).

To check whether the derivatized, imprinted (ISA / impr Chy) or the cross-linked, imprinted chymotrypsin (VIP) had the imprinted property in the organic solvent system equal to the native, imprinted chymotrypsin (na / impr Chy), all three imprint polypeptides were used for the synthesis of N-Ac- (D) -TrpEE tested in cyclohexane ( Fig. 5; analysis with HPLC, see Ståhl et al. 1991). It became apparent that the ISA / impr Chy and VIP had better activities than the na / impr Chy in the first 100 hours. The kinetic effects after 100 hours of reaction are due to the influence of the water released in the enzymatic condensation reaction in the organic solvent.

Example 4 Use of the cross-linked imprint polypeptide (VIP) in an aqueous medium

The VIP is suspended in phosphate buffer (10 mM), pH 7.8 and at approx. 10 min at 27 ° C pre-incubated in a thermal shaker. The reaction is carried out by adding the substrate, the N-Acetyl- (D) -tryptophanethylester (10 mM) started. The VIP showed a specific (D) - Ester hydrolysis activity of 0.04 µmol / (min · g). The native non-printed, not that cross-linked imprinted and the non-imprinted cross-linked α-chymotrypsin had none (D) Ester hydrolysis activity.

Example 5 Use of the cross-linked imprint polypeptide (VIP) in the organic solvent Pre-incubation (1h) in an aqueous solvent system

A. The VIP was incubated for 1 h in a phosphate buffer (10 mM), pH 7.8, at 25 ° C. After the batch had been lyophilized, the VIP was suspended in cyclohexane. After adding N-Ac- (D) -Trp (10 mM) and 20% (v / v) ethanol became the N-Acetyl- (D) -tryptophanethylester -Synthesis started at 25 ° C in a thermal shaker. The VIP showed a specific one (D) -Ester hydrolysis activity of 0.02 µmol / (min · g). The imprinted property did not work lost in the aqueous solvent system.

B. As in Example 5A. described, but using N-Ac- (D) -tyrosine instead of N-Ac- (D) -Trp.

C. As in Example 5A. described, but using N-Ac- (D) -phenylalanine instead of N-Ac- (D) -Trp.

Example 6 Production of a fixed, imprint polypeptide for selective adsorption

50 mg bovine serum albumin are dissolved in 10 ml 0.05 M potassium phosphate buffer pH 5.5. Itaconic anhydride is then added at room temperature with stirring added. The decrease in the pH value of the anhydride that occurs with each addition The reaction solution is corrected by adding 5 M NaOH. After the addition is complete of the anhydride is stirred for a further 1 hour and the pH is corrected again. The received Reaction solution is used to separate the low-molecular components using PD  10 gel filtration columns (Pharmacia) buffered in 0.05 M potassium phosphate buffer pH 5.5 and finally lyophilized.

The extent of the modification of the functional groups is included 2,4,6-trinitrobenzenesulfonic acid (TNBS assay).

30 mg of bovine serum albumin derivatized with itaconic anhydride (extent of Modification = 85%) are distilled in 4 ml of H₂O. solved. The aqueous solution contains L-malic acid in a concentration of 0.5 M. The solution is cooled at pH 5.5 to 0 ° C and 4 ml of 1-propanol, which was cooled to -20 ° C., were added. The resulting precipitate will centrifuged, washed with 10 ml of 1-propanol and finally lyophilized.

340 mg of ethylene glycol dimethacrylate with 4 mg of 2,2′-azo-bis- (2- methlypropiononitrile) dissolved. 10 mg of lyophilisate are suspended in it. The Copolymerization is initiated using UV light. The copolymerization time is 4 Hours. The covalently cross-linked and imprinted bovine serum albumin is made with cyclohexane washed and lyophilized.

As with Dabulis and Klibanov, Biotechnol. Bioeng. 39: 176-185 (1991) the extent of the binding affinity of the imprinted bovine serum albumin to L-malic acid examined. It was shown that both the imprinted and the networked, imprinted Bovine serum albumin in ethyl acetate had binding affinity for L-malic acid. In watery The medium was only the cross-linked, imprinted bovine serum albumin capable of L-malic acid to selectively adsorb.

Example 7 Preparation of a native, fixed polypeptide

In 29 ml of 0.05 M citric acid-phosphate buffer pH 6.0, 50 mg of 98% derivatized β-glucosidase (see Table 2) and 250 mg of N, N'-methylenebisacrylamide dissolved. The copolymerization is carried out by adding 0.5 ml of an ammonium peroxodisulfate solution (5% w / v) and 0.5 ml of a 3-dimethylaminopropionitrile solution (5% v / v) started. After a Response time of 5 hours, the copolymer is initially in a pleated filter with 0.5 Liter of 0.5 M NaCl solution and then with 2 liters of H₂O dest. washed until salt-free. The Copolymer is taken up in 0.05 M citric acid-phosphate buffer pH 6.0 and then lyophilized.

The enormous stabilization of the cross-linked polypeptide against the native, non-cross-linked is shown in Table 4 , in which the half-lives of the polypeptide are given at different temperatures.

Table 4

Half-lives of the catalytically active conformation of the free and cross-linked β-glucosidase

literature

Dabulis, K. and Klibanow, A. (1992). Biotechnol. Bioeng. 39, 176-185.
Fritz, H., Neudecker, M., Schult, H. and Werle, E. (1966). Applied Chem. 78, 775.
Goldstein, L. (1970). Methods Enzymol. 19, 935-962.
Habeeb, AFSA (1966). Anal. Biochem. 14, 328-336.
Kölle, R. (1995) Dissertation, TU Braunschweig, Germany, 53-59.
Mosbach, K. and Ramström, O. (1996). Bio / Technology 14, 163-170.
Russell, AJ and Klibanow, AM (1988). J Biol. Chem. 263, 11624-11626.
Ståhl, M., Jeppson-Wistrand, U., Mãnsson, M.-O. and Mosbach, K. (1991). J. Am. Chem. Soc. 113, 9366-9368.
Ståhl, M., Månsson, M.-O. and Mosbach, K. (1990). Biotechnol. Lett. 12, 161-166.

Claims (19)

1. A process for the preparation of covalently cross-linked imprint polypeptides which are fixed and stabilized in their conformation and which are stable in both aqueous and organic solvent systems, comprising the following steps:

• (A) covalently introducing polymerizable groups containing unsaturated bonds into a polypeptide;
• (B) imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium;
• (C) precipitation of the polypeptide / print molecule mixture obtained in step (B) by adding an additive suitable for precipitation and / or freeze-drying the polypeptide / print molecule mixture obtained in step (B); and
• (D) copolymerizing the imprint polypeptide obtained in step (C) in an organic solvent with a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.

2. The method according to claim 1, characterized in that step (B) before step (A) is carried out. 3. The method according to claim 1, characterized in that steps (A) and (B) be carried out simultaneously. 4. The method according to any one of the preceding claims, characterized in that a Polypeptide with OH, and / or NH₂, and / or SH groups is used, the partially or completely with the polymerizable, containing unsaturated bonds Compounds are derivatized to form covalent bonds. 5. The method according to any one of the preceding claims, characterized in that a native protein, especially an enzyme, is used as the polypeptide. 6. The method according to any one of the preceding claims, characterized in that the compounds containing unsaturated bonds used in step (A) or mixtures thereof are selected from the group consisting of reactive analogs or derivatives of the general formula (I) wherein the radicals R₁, R₂, R₃ and R₄ independently of one another hydrogen atoms, carboxyl radicals, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl radicals, alkoxy radicals or carboxyalkyl radicals with preferably up to 10, more preferably up to 6, in particular 1 or 2, carbon atoms or substituted or are unsubstituted aryl radicals or carboxyaryl radicals, with the proviso that at least one of the radicals R₁, R₂, R₃ and R₄ is independently a carboxyl, carboxyalkyl radical or carboxyaryl radical. 7. The method according to claim 6, characterized in that the used in step (A) compounds containing unsaturated bonds are selected from the group consisting of itaconic, maleic, citracon, croton, methacrylic, acrylic acid and their Anhydrides and acid halides. 8. The method according to any one of the preceding claims, characterized in that in Step (A) additionally polyols and / or salts are added. 9. The method according to any one of the preceding claims, characterized in that the crosslinker used for the copolymerization in step (D) is selected from the group consisting of analogs or derivatives of the general formula (II) H₂C = C (R₁) - (R₂) _n -C (R₃) = CH₂ (II) where the radicals R₁ and R₃ independently of one another are hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxyl, carboxyalkyl or alkyl ether radicals, preferably with up to 10, in particular 1 or 2 carbon atoms or substituted or unsubstituted aryl radicals and the radical R₂ is a cyclic or linear, saturated or unsaturated, substituted or unsubstituted alkyl or alkyl ether radical with preferably up to 10, in particular 1 or 2 carbon atoms or an aryl radical and n has a value of preferably up to to 10 and especially 1 or 2. 10. The method according to claim 9, characterized in that the copolymerization in Step (D) used crosslinker is selected from the group consisting of Divinylalkyl and divinylaryl compounds.   11. The method according to claim 1 to 8, characterized in that the crosslinker used for the copolymerization in step (D) is selected from the group consisting of analogs or derivatives of the general formula (III) wherein the radicals R₁, R₂ and R₃ and n have the meaning given in claim 9 for the general formula (II) and the radical X is an oxygen atom or the group -NH. 12. The method according to claim 11, characterized in that the copolymerization in Step (D) used crosslinker is selected from the group consisting of Ethylene glycol dimethacrylate, N, N'-methylene-bis-acrylamide, diitaconic acid alkyl amides and diitaconic acid arylamides. 13. A method for producing a covalently cross-linked imprint polypeptide which is fixed and stabilized in its conformation and which is stable in both aqueous and organic solvent systems, comprising the following steps:

• (A) covalently introducing polymerizable groups containing unsaturated bonds into a polypeptide in an aqueous medium;
• (B) imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium; and
• (C) copolymerization of the polypeptide obtained in step (B) in an aqueous medium with a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.

14. The method according to claim 13, characterized in that step (B) before step (A) is carried out. 15. The method according to claim 13, characterized in that steps (A) and (B) be carried out simultaneously. 16. Fixed and stabilized in its conformation, covalently networked Imprint polypeptide that is stable in both aqueous and organic solvent systems is obtainable by a method according to any one of the preceding claims. 17. Use of a polypeptide according to claim 16 in catalytic, chromatographic and / or analytical methods in aqueous or organic Solvent systems are carried out.   18. Use of a polypeptide according to claim 16 for catalytic synthesis, Chromatography and analysis of chiral compounds. 19. Use of a polypeptide according to claim 16 in biosensor technology for specific recognition of molecules.