EP1326880A1 - Method for the selective modification of peptides and proteins - Google Patents
Method for the selective modification of peptides and proteinsInfo
- Publication number
- EP1326880A1 EP1326880A1 EP01982347A EP01982347A EP1326880A1 EP 1326880 A1 EP1326880 A1 EP 1326880A1 EP 01982347 A EP01982347 A EP 01982347A EP 01982347 A EP01982347 A EP 01982347A EP 1326880 A1 EP1326880 A1 EP 1326880A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- proteins
- peptides
- ala
- peptidases
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000012982 x-ray structure analysis Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
Definitions
- the invention relates to a method for the region-specific modification of peptides and proteins with probes and reporter molecules using biocatalysts.
- N-terminal ⁇ -amino groups are preferred targets for selective modifications
- the ⁇ -amino groups ubiquitously in lysine residues occurring in proteins and peptides do not allow targeted introduction of marker and reporter groups, but also other derivatizations, e.g. Pegylation, at the N-terminus.
- Chemical acylation reactions are carried out with anhydrides or primarily with active esters, e.g. ⁇ / -Hydroxysuccinimidest- or 4-nitrophenyl esters carried out, with which other side chain functions of proteinogenic amino acid residues can also react and thus rule out a selective / ⁇ - modification.
- the object of the invention is the regiospecific biocatalytic modification of peptides and proteins at the N-terminus while excluding side reactions as completely as possible.
- a peptidase being the biocatalyst is used in conjunction with a non-amino acid or non-peptide-like substrate mimetic.
- substrate mimetic was coined by Bordusa et al., Angew. Chem. 109 (1997), 2583-2585 and Bordusa et al., Angewandte Chemie International Edition in English, 36 (1997), 2473-2475.
- Leaving group is known to the person skilled in the art and is explained by F. Bordusa, Braz. J. Med. Biol. Res. (See above) (in particular in FIG. 1).
- the N-terminal biocatalytic modification of a peptide or protein takes place contrary to the prevailing opinion of the professional world with peptidases, the non-amino acid-like or non-peptide-like grouping to be introduced in the form of an ester derivative carrying a leaving group which carries the native specificity of the one used by targeted manipulation Turns off enzyme, thereby enabling the catalysis of an irreversible ⁇ ⁇ acylation.
- the theoretical basis, the proposed reaction mechanism and the preparation of substrate mimetics for various proteases and peptidases is described in the review article by F. Bordusa, Brazilian Journal of Medical and Biological Research 33 (2000), 469-485.
- the results according to the invention are very surprising.
- a marker or reporter group all possible marker or Reporter groups are used, for example aminobenzyl, phlorethyl, biotinyl groups.
- marker groups can be used which are required for the diagnostic use of the peptides or proteins, such as haptens (biotin, digoxin, digoxigenin, digitoxin etc.) or labels (dyes, radioactively labeled compounds, fluorescent groups, electrochemiluminescent labels (Elecsys), luminophores , Etc.).
- Substances that change or improve the properties of proteins, such as solubility, etc. can also be selected as the marker or reporter group.
- substances such as polyethylene glycol (PEG) and derivatives thereof can be used to optimize the properties of proteins or peptides, such as erythropoietin, insulin, monoclonal antibodies or other therapeutically active proteins and peptides. Examples of such therapeutic proteins and peptides and substances for optimizing the therapeutic effectiveness are known to the person skilled in the art.
- Organic-chemical ester derivatives whose acyl residues correspond to the marker or reporter groups to be introduced and whose leaving groups carry specificity determinants of selected serine or cvstein peptidases are preferably used as reagents for the biocatalytic ⁇ T acylations according to the invention.
- the terms leaving group and specificity determinants are known to the person skilled in the art (see, for example, F. Bordusa, Braz. J. Med. Biol. Res. (See above)).
- the leaving group of a substrate mimetic binds in place of the specificity-mediating amino acid side chain of the normal substrate (Thormann et al., Biochemistry 38 (1999), 6056-6062).
- ⁇ T-selective modifications of peptides and proteins are preferably achieved by using a carboxylic acid derivative whose carboxyl function which reacts is present as an ester with a specificity determinant in the leaving group which corresponds to the peptidase used, and a peptide or protein to be labeled in which the Reacting ⁇ -amino function is unblocked, in the presence of the corresponding peptidase, in solution at room temperature, or also in the frozen state or at low temperatures.
- Suitable peptidases are trypsin, chymotrypsin, V8 protease, glu-specific endopeptidase from Bacillus licheniformis, subtilisin, mutants of these enzymes such as, for example, the trypsin mutant trypsin D189K + K60E (preparation see Example 9) or enzymes with similar specificity determinants.
- the non-compliant term peptidase was used instead of proteases.
- modified peptides and proteins can be separated and purified using conventional methods in protein chemistry.
- Example 1 V8 protease-catalyzed N-terminal introduction of 2-aminobenzoic acid into peptides
- 2-aminobenzoic acid carboxymethylthioester hereinafter referred to as 2-ABz-SCm
- 2-ABz-SCm 2-aminobenzoic acid carboxymethylthioester
- 2-ABz-SCm and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively.
- An aqueous buffer system with a small proportion of organic solvent was used as the solvent.
- the reaction was started by adding the enzyme and, after virtually complete conversion of 2-ABz-SCm, ended by inactivating the enzyme.
- the analysis and quantification of the reaction was carried out using chromatographic methods.
- the enzyme catalysis led to a 99% conversion of Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly into the corresponding N-terminal 2-ABz-modified analogues.
- the identity of the synthesis product was checked by the usual methods of organic chemistry.
- the carboxy component used was phloretyl-carboxymethylthioester, hereinafter referred to as phloretyl-SCm
- the amino component was the decapeptide Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly.
- Phloretyl-SCm and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively.
- An aqueous buffer system with a small proportion of organic solvent was used as the solvent.
- the reaction was started by adding the enzyme and, after virtually complete conversion of phloretyl-SCm, ended by inactivating the enzyme.
- 2-aminobenzoic acid, 4-guanidinophenyl ester was designated as the carboxy component, hereinafter referred to as 2-ABz-OGp, and the oligopeptide Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys as the amino component -Ala-Ala- Gly-Ala-Tyr used.
- 2-ABz-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively.
- An aqueous buffer system with a small proportion of organic solvent was used as the solvent.
- the reaction was started by adding the enzyme and, after virtually complete conversion of 2-ABz-OGp, ended by inactivating the enzyme.
- the analysis and quantification of the reaction was carried out using chromatographic methods.
- the enzyme catalysis led to a 98.8% conversion of Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr into the corresponding N-terminal 2 -ABz-modified analogues.
- the identity of the synthesis product was checked by the usual methods of organic chemistry.
- the reaction neither led to a modification of trifunctional side chains, nor to a detectable proteolytic cleavage.
- the carboxy component was phloretyl-4-guanidinophenyl ester, hereinafter referred to as phloretyl-OGp
- the amino component was the oligopeptide Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala - Gly-Ala-Tyr used.
- Phloretyl-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively.
- An aqueous buffer system with a small proportion of was used as the solvent organic solvent.
- the reaction was started by adding the enzyme and, after virtually complete conversion of phloretyl-OGp, ended by inactivating the enzyme.
- the analysis and quantification of the reaction was carried out using chromatographic methods. Enzyme catalysis led to a 99.3% conversion of Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly into the corresponding N-terminal phloretyl-modified analogues.
- the identity of the synthesis product was checked by the usual methods of organic chemistry. The reaction neither led to a modification of trifunctional side chains, nor to a detectable proteolytic cleavage.
- 2-aminobenzoic acid-4-guanidinophenyl ester hereinafter referred to as 2-ABz-OGp
- 2-ABz-OGp 2-aminobenzoic acid-4-guanidinophenyl ester
- 2-ABz-OGp 2-aminobenzoic acid-4-guanidinophenyl ester
- 2-ABz-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively.
- An aqueous buffer system with a small proportion of organic solvent was used as the solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of 2-ABz-OGp, ended by inactivating the enzyme.
- the analysis and quantification of the reaction was carried out using chromatographic methods.
- the enzyme catalysis led to a 94.4% conversion of Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly into the corresponding N-terminal 2-ABz-modified analogues.
- the identity of the synthesis product was checked by the usual methods of organic chemistry.
- the carboxy component was phloretyl-4-guanidinophenyl ester, hereinafter referred to as phloretyl-OGp
- the amino component was the decapeptide Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly used.
- Phloretyl-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively.
- An aqueous buffer system with a small proportion of organic solvent was used as the solvent.
- the reaction was started by adding the enzyme and, after virtually complete conversion of phloretyl-OGp, ended by inactivating the enzyme.
- the carboxyl component was BiotinyI-4-guanidinophenyl ester, hereinafter referred to as Biotinyl-OGp, and the protein E. coli Parvulin 10 was used as the amino component.
- Biotinyl-OGp and parvulin were used in a ratio of 1: 4 in a concentration of 2mM and 8mM, respectively.
- An aqueous buffer system with a small proportion of an organic solvent was used as the solvent.
- 0.1 M HEPES N 2 - [2-hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid
- buffer pH 8.0 0.1 M NaCl, 0.01 M CaCl 2 and 8% (v / v) DMF (dimethylformamide) used.
- the reaction was started by adding the enzyme trypsin D189K + K60E (trypsin mutant in which the amino acid D at position 189 has been replaced by K or K at position 60 by E; preparation see Example 9) and ended after a reaction time of 2 hours.
- the enzyme was used in a concentration of 6.5 x 10 "6 M.
- the analysis and quantification of the reaction was carried out by MALDI-MS spectroscopy (FIG. 1).
- the enzyme catalysis led to a conversion of E. coli parvulin 10 into the N- terminally biotinylated biotinyl (E. coli parvulin 10).
- E. coli parvulin 10 The primary sequence of E. coli parvulin 10 is known and corresponds to the following amino acid sequence.
- AKTAAALHIL VKEEKLALDL LEQIKNGADF GKLAKKHSIC PSGKRGGDLG EFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RN
- the protein RNase T1 was used as the carboxyl component biotinyl-OGp and as the amino component.
- a variant with an additional Arg (R) -Gly (G) residue at the N-terminus of the protein was used for the biotinylation of RNase T1.
- RNase T1 variant RG-RNase T1
- G-RNase T1 and the wild type RNase T1 were also obtained. This mixture was used without separation of the individual variants for the enzyme-catalyzed biotinylation.
- Biotinyl-OGp and RNase T1 mixture were used in a ratio of 1: 4 in a concentration of 2 mM and 8 mM, respectively.
- An aqueous buffer system as described in Example 7 was used as the solvent.
- the reaction was started by adding the enzyme trypsin D189K + K60E (6.5 x 10 "6 M). The reaction time was 2 hours.
- the analysis and quantification of the reaction were carried out by chromatographic methods.
- the electropherogram of the capillary electrophoresis carried out is shown in FIG It can be clearly seen that the RG-RNase T1 was converted almost quantitatively into the Biotinyl-RG-RNase T1.
- the E. coli Vector pST was used to carry out the site-directed mutagenesis. This contains part of the Bluescript vector and the gene for anionic rat trypsin, which is fused to an ⁇ -factor leader and an ADH / GAPDH promoter. Protein expression was carried out using the pYT plasmid, a pBS24 derivative which carries selection markers for uracil- and leucine-deficient medium.
- Both the pST and pYT plasmids have an ampicillin resistance gene.
- the maps of both vectors, i.e. the plasmids pST (5.4 kb) and pYT (14 kb), with the corresponding interfaces, are shown in FIG. 3.
- the site-directed mutagenesis was carried out using the Quik change ® kit (STRATAGENE) in the E. coli plasmid pST.
- the method used is similar to a PCR, with two plasmid strands of the pST vector of PFU polymerase being replicated starting from two synthetic oligonucleotide primers which contain the desired mutation. Wild-type pST served as a template for generating single mutations. These single mutants were the starting point for the construction of the double mutant.
- the PCR product obtained was transformed into ultra-competent E. coli XL II blue cells (STRATAGENE). The subsequent selection was carried out on nutrient agar plates (LB-amp) containing ampicillin. The picked colonies were transferred to a liquid medium containing ampicillin (LB-amp) and, after culturing for one day, the plasmid was isolated using the SNAP kit (INVITROGENE). The isolated DNA was checked by electrophoresis with a 1% agarose gel. By sequencing the complete gene it was possible to ensure that only the desired mutations were contained.
- the yeast cell strain used is called Saccharomyces cerevisiae DLM 101 ⁇ [Mat a, leu 2-3, -112 to 2, 3-11, -15 can 1, ura 3 ⁇ , pep4 ⁇ , [cir °], DM 23].
- the EZ yeast transformation kit (ZYMO-RESEARCH) was used to produce competent yeast cells and transform the pYT plasmids. The selection was made on uracil-deficient SC plates by incubation at 30 ° C. for 3 to 4 days. Single colonies were further vaccinated on leucine-deficient SC plates and also incubated for 3 to 4 days at 30 ° C, which increased the number of copies of the plasmid in the cells.
- the cells were first separated by centrifugation for 20 min at 4000 rpm and the supernatant adjusted to pH 4.0 was centrifuged again at 12,000 rpm.
- the practically particle-free trypsinogen-containing supernatant was applied to a Toyopearl 650 M (SUPELCO) cation exchange column equilibrated with 2 mM sodium acetate / 100 mM acetic acid (pH 4.5). Elution was carried out using a linear pH gradient starting from 2 mM sodium acetate / 100 mM acetic acid (pH 4.5) to 200 mM Tris / HCl (pH 8.0).
- the trypsinogen-containing fractions could be determined and summarized by SDS-polyacrylamide gel electrophoresis using a 15% polyacrylamide gel.
- the volume of the protein solutions was reduced to about 10 to 15 ml using Centriprep concentrators (AMICON).
- the activation of the trypsinogen variant to the corresponding trypsin D189K + K60E was carried out using highly purified enterokinase (BIOZYME) at pH 6.5 and was monitored by SDS gel electrophoresis.
- BIOZYME highly purified enterokinase
- the activated enzyme was purified using a Biocad Sprint Perfusion Chromatography System (PERSEPTIVE BIOSYSTEMS).
- the protein samples were separated on a POR ⁇ S 20 HQ - anion exchange column (4 x 100 mm, PERSEPTIVE BIOSYSTEMS) equilibrated with 5% bis- / tris-propane pH 6.0 and subsequent gradient elution up to 95% 3M NaCI solution.
- the fractions containing trypsin were checked for purity using an SDS gel and pooled. Finally, dialysis was carried out against 1 mM HCI at 4 ° C and concentration of the samples with Centriprep concentrators to 2 to 4 ml.
- the final yields were about 2 to 5 mg protein per liter of culture medium.
- the protein concentration of the preparations was determined using the Bradford method on a spectrophotometer at a wavelength of 595 nm.
- the calibration curve was recorded using a series of bovine trypsin dilutions between 50 ⁇ m / ml and 1 mg / ml.
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Abstract
The invention relates to a method for the area-specific modification of peptides and proteins, using probes and reporter molecules with the help of peptidases coupled with a non-amino acid type or non-peptide type substrate mimetic.
Description
Verfahren zur selektiven Modifizierung von Peptiden und ProteinenProcess for the selective modification of peptides and proteins
Die Erfindung betrifft ein Verfahren zur regiospezifischen Modifizierung von Peptiden und Proteinen mit Sonden und Reportermolekülen unter der Verwendung von Biokatalysatoren.The invention relates to a method for the region-specific modification of peptides and proteins with probes and reporter molecules using biocatalysts.
Durch die Sequenzierung der Genome des Menschen und anderer Organismen resultiert eine enorme Flut von Proteinsequenzen, deren biochemische Funktion sehr häufig nur unzureichend oder überhaupt nicht aufgeklärt ist. Die Proteine sind funktionelle Genprodukte und daher für alle Aktivitäten der biologischen Welt verantwortlich. Aus diesem Grunde ist das Verstehen der Proteinstruktur und -funktion eine Notwendigkeit moderner biologischer Forschung (vgl. T.E. Creighton, Proteins. Structure and Molecular Properties, W.H. Freeman & Co. New York, 1993). Zur Aufklärung sind gezielte, schonende Markierungen mit speziellen Sonden und Reportergruppen essentiell, um die molekularen Prozesse in vitro und in vivo verfolgen zu können. Im Mittelpunkt stehen u.a. die Fluoreszenzmarkierung (vgl. R.P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc., 1996), die Einführung von Spinlabel (W.L. Hubbel, C. Alienbach, Investigations of Structure and Dynamics in Membrane Proteins Using Site-directed Spin Labeling, Curr. Opin. Struct. Biol. 4 (1994) 566-573), Photoaffinitätsmarkierung (D.I. Schuster, W.C. Probst, G.K. Ehrung, G. Singh, Photoaffinity Labeling, Photochemistry and Photobiology 49 (1988) 785-804) sowie die "Biotin-Technik" (M. Wilchek, E.A. Bayer, Avidin-Biotin Technology in Meth. Enzymol. V. 184, Academic Press, 1990).The sequencing of the genomes of humans and other organisms results in an enormous flood of protein sequences, the biochemical function of which is very often insufficiently or not at all explained. The proteins are functional gene products and therefore responsible for all activities in the biological world. For this reason, understanding protein structure and function is a necessity of modern biological research (see T.E. Creighton, Proteins. Structure and Molecular Properties, W.H. Freeman & Co. New York, 1993). Targeted, gentle markings with special probes and reporter groups are essential for clarification in order to be able to follow the molecular processes in vitro and in vivo. The focus is on fluorescent labeling (see RP Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc., 1996), the introduction of Spinlabel (WL Hubbel, C. Alienbach, Investigations of Structure and Dynamics in Membrane Proteins Using Site-directed Spin Labeling, Curr. Opin. Struct. Biol. 4 (1994) 566-573), photoaffinity labeling (DI Schuster, WC Probst, GK Ehrung, G. Singh, Photoaffinity Labeling, Photochemistry and Photobiology 49 (1988) 785-804) and die "Biotin technology" (M. Wilchek, EA Bayer, Avidin-Biotin Technology in Meth. Enzymol. V. 184, Academic Press, 1990).
Zur Modifizierung von Peptiden und Proteinen spielten - und spielen noch immer - chemische Verfahren (vgl. T. Imoto, H. Yamada, Chemical Modification, in Protein Function. A Practical Approach (T.E. Creighton, ed.) pp. 247-277, IRL Press, 1989; G.E. Means, R.E. Feeney, Chemical Modification of Proteins, Holden-Day, 1971 ) eine bedeutende Rolle in der Proteinforschung. So ist trotz des schnellen Fortschritts der NMR-Technik, die im letzten Jahrzehnt eine vollständige Signalzuordnung und somit eine Aufklärung der 3D-Strukturen von Proteinen bis zu 150 - 200 Aminosäurebausteinen ermöglichte, die chemische Modifizierung weiterhin auch ein
Werkzeug zur Raumstrukturbestimmung in Lösung, da große Proteine der NMR- Strukturanalyse nicht zugänglich sind und die Röntgenstrukturanalyse Proteinkristalle erfordert, die in sehr vielen Fällen nicht erhalten werden können.Chemical processes played (and still play) to modify peptides and proteins (cf. T. Imoto, H. Yamada, Chemical Modification, in Protein Function. A Practical Approach (TE Creighton, ed.) Pp. 247-277, IRL Press, 1989; GE Means, RE Feeney, Chemical Modification of Proteins, Holden-Day, 1971) play an important role in protein research. Despite the rapid progress of NMR technology, which has enabled complete signal assignment and thus elucidation of the 3D structures of proteins of up to 150-200 amino acid units in the past decade, chemical modification is still possible Tool for spatial structure determination in solution, since large proteins are not accessible for NMR structure analysis and X-ray structure analysis requires protein crystals, which in many cases cannot be obtained.
Da N-terminale α-Aminogruppen bevorzugte Ziele von selektiven Modifizierungen sind, erlauben die ε-Aminogruppen ubiquitär in Proteinen und Peptiden vorkommender Lysinreste keine gezielte Einführung von Marker- und Reportergruppen aber auch andere Derivatisierungen, wie z.B. Pegylierung, an den N-Terminus. Chemische Acylierungsreaktionen werden mit Anhydriden oder vorrangig mit aktiven Estern, wie z.B. Λ/-Hydroxysuccinimid- oder 4-Nitrophenylestem durchgeführt, womit aber auch andere Seitenkettenfunktionen proteinogener Aminosäurereste reagieren können und damit eine selektive /^-Modifikation ausschließen. Lediglich der Phenylacetyl-Rest wurde spezifitätsdeterminiert durch die Penicillin-Acylase in Umkehrung der nativen Wirkung als Schutzgruppe für Aminosäuren im Rahmen von Peptidsynthesen enzymatisch eingeführt (R. Didziapetris, B. Drabnig, V. Schellenberger, H.-D. Jakubke, V. Svedas, FEBS Lett. 287 (1991) 31-33) und durch das gleiche Enzym wieder abgespalten (vgl. Review: A. Reidel, H. Waldmann, J. prakt. Chem. 335 (1993) 109- 127). Abgesehen von dieser direkten Schutzgruppeneinführung wurden nur solche Methoden beschrieben, die auf einer Übertragung bereits N-terminal markierter Aminosäure- oder Peptidderivate mit peptidasespezifischen Aminosäureresten in der PrPosition unter der Katalyse von Peptidasen beruhen und zwangsläufig keine Irreversibilität aufweisen. Das für CN-Ligationen von Peptid- und Proteinsegmenten entwickelte Substratmimetika-Konzept (F. Bordusa, D. Ulimann, C. Eisner, H.-D. Jakubke, Angew. Chem. 109 (1997) 2583-25-85; Review: F. Bordusa, Braz. J. Med. Biol. Res. 72 (2000) 469-485) hat dagegen den Vorteil der Irreversibilität.Since N-terminal α-amino groups are preferred targets for selective modifications, the ε-amino groups ubiquitously in lysine residues occurring in proteins and peptides do not allow targeted introduction of marker and reporter groups, but also other derivatizations, e.g. Pegylation, at the N-terminus. Chemical acylation reactions are carried out with anhydrides or primarily with active esters, e.g. Λ / -Hydroxysuccinimidest- or 4-nitrophenyl esters carried out, with which other side chain functions of proteinogenic amino acid residues can also react and thus rule out a selective / ^ - modification. Only the phenylacetyl residue was enzymatically determined by penicillin acylase in reverse of the native effect as a protective group for amino acids in the context of peptide synthesis (R. Didziapetris, B. Drabnig, V. Schellenberger, H.-D. Jakubke, V. Svedas , FEBS Lett. 287 (1991) 31-33) and split off again by the same enzyme (cf. Review: A. Reidel, H. Waldmann, J. Prakt. Chem. 335 (1993) 109-127). Apart from this direct introduction of protective groups, only those methods have been described which are based on a transfer of already N-terminally labeled amino acid or peptide derivatives with peptidase-specific amino acid residues in the preposition under the catalysis of peptidases and inevitably have no irreversibility. The substrate mimetic concept developed for CN ligations of peptide and protein segments (F. Bordusa, D. Ulimann, C. Eisner, H.-D. Jakubke, Angew. Chem. 109 (1997) 2583-25-85; Review: F. Bordusa, Braz. J. Med. Biol. Res. 72 (2000) 469-485), on the other hand, has the advantage of irreversibility.
Aufgabe der Erfindung ist die regiospezifische biokatalytische Modifizierung von Peptiden und Proteinen am N-Terminus unter möglichst vollständigem Ausschluß von Nebenreaktionen.The object of the invention is the regiospecific biocatalytic modification of peptides and proteins at the N-terminus while excluding side reactions as completely as possible.
Diese Aufgabe wird gelöst mit einem Verfahren zur selektiven biokatalytischen Modifizierung von Peptiden und/oder Proteinen, wobei als Biokatalysator eine Peptidase
in Verbindung mit einem nicht-aminosäure- oder nicht-peptidartigen Substratmimetikum eingesetzt wird. Der Begriff „Substratmimetikum" wurde von Bordusa et al., Angew. Chem. 109 (1997), 2583-2585 und Bordusa et al., Angewandte Chemie International Edition in Englisch, 36 (1997), 2473-2475 geprägt. Der Begriff der Abgangsgruppe ist dem Fachmann bekannt und wird bei F. Bordusa, Braz. J. Med. Biol. Res. (siehe oben) erklärt (insbesondere in der Figur 1 ).This object is achieved with a method for the selective biocatalytic modification of peptides and / or proteins, a peptidase being the biocatalyst is used in conjunction with a non-amino acid or non-peptide-like substrate mimetic. The term "substrate mimetic" was coined by Bordusa et al., Angew. Chem. 109 (1997), 2583-2585 and Bordusa et al., Angewandte Chemie International Edition in English, 36 (1997), 2473-2475. The term " Leaving group is known to the person skilled in the art and is explained by F. Bordusa, Braz. J. Med. Biol. Res. (See above) (in particular in FIG. 1).
Gemäß der vorliegenden Erfindung erfolgt die N-terminale biokatalytische Modifizierung eines Peptids oder Proteins entgegen der vorherrschenden Meinung der Fachwelt mit Peptidasen, wobei durch eine gezielte Manipulation die einzuführende nichtaminosäureartige bzw. nichtpeptidartige Gruppierung in Form eines Esterderivates eine Abgangsgruppe trägt, die die native Spezifität des eingesetzten Enzyms ausschaltet, und dadurch die Katalyse einer irreversiblen Λ^-Acylierung ermöglicht. Die theoretischen Grundlagen, der postulierte Reaktionsmechanismus sowie die Herstellung von Substratmimetika für verschiedene Proteasen und Peptidasen, wird in dem Übersichtsartikel von F. Bordusa, Brazilian Journal of Medical and Biological Research 33 (2000), 469-485 beschrieben. Im Gegensatz zu chemischen Acylierungsreaktionen werden aufgrund der Regiospezifität von Peptidasen reaktionsfähige Seitenkettenfunktionen von trifunktionellen Aminosäurebausteinen in den zu modifizierenden Peptiden und Proteinen nicht acyliert, wodurch eine absolut selektive Einführung von Marker- und Reportergruppen an die Λ^-Aminogruppe des entsprechenden Peptids oder Proteins garantiert wird. Weiterhin muß die für die Modifizierung vorgesehene Gruppierung nicht bereits an einem enzymatisch anzuknüpfenden Aminosäure- oder Peptid-Rest gebunden sein, wie es bei einigen literaturbekannten Verfahren eine notwendige Voraussetzung ist und die Gefahr einer reversiblen Spaltung in sich birgt.According to the present invention, the N-terminal biocatalytic modification of a peptide or protein takes place contrary to the prevailing opinion of the professional world with peptidases, the non-amino acid-like or non-peptide-like grouping to be introduced in the form of an ester derivative carrying a leaving group which carries the native specificity of the one used by targeted manipulation Turns off enzyme, thereby enabling the catalysis of an irreversible Λ ^ acylation. The theoretical basis, the proposed reaction mechanism and the preparation of substrate mimetics for various proteases and peptidases is described in the review article by F. Bordusa, Brazilian Journal of Medical and Biological Research 33 (2000), 469-485. In contrast to chemical acylation reactions, due to the regiospecificity of peptidases, reactive side chain functions of trifunctional amino acid units in the peptides and proteins to be modified are not acylated, which guarantees an absolutely selective introduction of marker and reporter groups to the Λ ^ amino group of the corresponding peptide or protein. Furthermore, the grouping provided for the modification need not already be bound to an amino acid or peptide residue to be linked enzymatically, as is a necessary prerequisite in some processes known from the literature and involves the risk of reversible cleavage.
Da nach der auf dem erfindungsgemäßen Wege erfolgten biokatalytischen Einführung der Marker- oder Reportergruppe die eingesetzte Peptidase eine derartig substituierte Amidbindung nicht mehr als Substrat erkennt und somit eine reversible enzymatische Abspaltung ausgeschlossen wird, sind die erfindungsgemäßen Ergebnisse sehr überraschend. Als Marker- oder Reportergruppe können alle möglichen Marker- oder
Reportergruppen Verwendung finden, beispielsweise Aminobenzyl-, Phlorethyl-, Biotinyl-Gruppen. Insbesondere können Markergruppen verwendet werden, die bei der diagnostischen Verwendung der Peptide oder Proteine benötigt werden, wie Haptene (Biotin, Digoxin, Digoxigenin, Digitoxin etc.) oder Label (Farbstoffe, radioaktiv markierte Verbindungen, Fluoreszenzgruppen, Elektrochemilumineszenz-Label (Elecsys), Luminophore, etc.). Als Marker- oder Reportergruppe können auch Substanzen gewählt werden, die die Eigenschaften von Proteinen, wie Löslichkeit etc., verändern oder verbessern. Insbesondere können Substanzen, wie Polyethylenglycol (PEG) sowie Derivate hiervon, Verwendung finden, um die Eigenschaften von Proteinen oder Peptiden, wie beispielsweise Erythropoietin, Insulin, monoklonale Antikörper oder andere therapeutisch wirksame Proteine und Peptide, zu optimieren. Beispiele für solche therapeutischen Proteine und Peptide sowie Substanzen zur Optimierung der therapeutischen Wirksamkeit sind dem Fachmann bekannt.Since after the biocatalytic introduction of the marker or reporter group in the way according to the invention, the peptidase used no longer recognizes such a substituted amide bond as a substrate and thus a reversible enzymatic cleavage is excluded, the results according to the invention are very surprising. As a marker or reporter group, all possible marker or Reporter groups are used, for example aminobenzyl, phlorethyl, biotinyl groups. In particular, marker groups can be used which are required for the diagnostic use of the peptides or proteins, such as haptens (biotin, digoxin, digoxigenin, digitoxin etc.) or labels (dyes, radioactively labeled compounds, fluorescent groups, electrochemiluminescent labels (Elecsys), luminophores , Etc.). Substances that change or improve the properties of proteins, such as solubility, etc., can also be selected as the marker or reporter group. In particular, substances such as polyethylene glycol (PEG) and derivatives thereof can be used to optimize the properties of proteins or peptides, such as erythropoietin, insulin, monoclonal antibodies or other therapeutically active proteins and peptides. Examples of such therapeutic proteins and peptides and substances for optimizing the therapeutic effectiveness are known to the person skilled in the art.
Vorzugsweise werden für die erfindungsgemäßen biokatalytischen ΛT-Acylierungen als Reagenzien organisch-chemische Esterderivate verwendet, deren Acylreste den einzuführenden Marker- oder Reportergruppen entsprechen und deren Abgangsgruppen Spezifitätsdeterminanten ausgewählter Serin- oder Cvsteinpeptidasen tragen. Die Begriffe Abgangsgruppe sowie Spezifitätsdeterminanten sind dem Fachmann bekannt (siehe beispielsweise F. Bordusa, Braz. J. Med. Biol. Res. (siehe oben)). Wie bei F. Bordusa beschrieben, bindet die Abgangsgruppe eines Substratmimetikums an Stelle der Spezifität-vermittelnden Aminosäureseitenkette des normalen Substrates (Thormann et al., Biochemistry 38 (1999), 6056-6062). Eine wichtige Eigenschaft der Substratmimetika ist daher die hohe Affinität der Abgangsgruppe zur primären Substratspezifität des jeweiligen Enzyms, z.B. zur starken Glu-Präferenz der V8-Protease an der Si-Stelle des katalytischen Zentrums. Das Auffinden, Austesten und Optimieren solcher Abgangsgruppen für Substratmimetika wird bei F. Bordusa, Braz. J. Med. Biol. Res. (siehe oben) beschrieben.Organic-chemical ester derivatives whose acyl residues correspond to the marker or reporter groups to be introduced and whose leaving groups carry specificity determinants of selected serine or cvstein peptidases are preferably used as reagents for the biocatalytic ΛT acylations according to the invention. The terms leaving group and specificity determinants are known to the person skilled in the art (see, for example, F. Bordusa, Braz. J. Med. Biol. Res. (See above)). As described by F. Bordusa, the leaving group of a substrate mimetic binds in place of the specificity-mediating amino acid side chain of the normal substrate (Thormann et al., Biochemistry 38 (1999), 6056-6062). An important property of substrate mimetics is therefore the high affinity of the leaving group for the primary substrate specificity of the respective enzyme, e.g. on the strong Glu preference of the V8 protease at the Si site of the catalytic center. Finding, testing and optimizing such leaving groups for substrate mimetics is described in F. Bordusa, Braz. J. Med. Biol. Res. (See above).
Die praktizierte Verfahrensweise, d.h. die Auswahl und Synthese der für die enzymatische Λf-Acylierung eingesetzten Substrate in Form von Carbonsäureestern,
die Wahl des Puffersystems, der Reaktionszeit u.a. ist verhältnismäßig unkritisch und kann vom Fachmann für enzymatische Transformationen einfach ermittelt werden.The procedure practiced, ie the selection and synthesis of the substrates used for the enzymatic Λf acylation in the form of carboxylic acid esters, the choice of the buffer system, the reaction time and others is relatively uncritical and can be easily determined by a person skilled in the art for enzymatic transformations.
Erfindungsgemäß werden ΛT-selektive Modifikationen von Peptiden und Proteinen bevorzugt erreicht durch Verwendung eines Carbonsäurederivates, dessen in Reaktion tretende Carboxylfunktion als Ester mit einer Spezifitätsdeterminante in der Abgangsgruppe vorliegt, die der eingesetzten Peptidase entspricht, und einem zu markierenden Peptid oder Protein, bei dem die in Reaktion tretende α-Aminofunktion unblockiert ist, in Gegenwart der entsprechenden Peptidase, in Lösung bei Raumtemperatur, oder auch im gefrorenen Zustand bzw. bei tiefen Temperaturen. Als Peptidasen kommen beispielsweise Trypsin, Chymotrypsin, V8 Protease, Glu- spezifische Endopeptidase aus Bacillus licheniformis, Subtilisin, Mutanten dieser Enzyme wie beispielsweise die Trypsinmutante Trypsin D189K + K60E (Herstellung siehe Beispiel 9) oder Enzyme mit ähnlichen Spezifitätsdeterminanten in Frage. In der vorliegenden Beschreibung wurde der nonemklaturgerechte Terminus Peptidase anstelle von Proteasen verwendet.According to the invention, ΛT-selective modifications of peptides and proteins are preferably achieved by using a carboxylic acid derivative whose carboxyl function which reacts is present as an ester with a specificity determinant in the leaving group which corresponds to the peptidase used, and a peptide or protein to be labeled in which the Reacting α-amino function is unblocked, in the presence of the corresponding peptidase, in solution at room temperature, or also in the frozen state or at low temperatures. Examples of suitable peptidases are trypsin, chymotrypsin, V8 protease, glu-specific endopeptidase from Bacillus licheniformis, subtilisin, mutants of these enzymes such as, for example, the trypsin mutant trypsin D189K + K60E (preparation see Example 9) or enzymes with similar specificity determinants. In the present description, the non-compliant term peptidase was used instead of proteases.
Befinden sich in dem zu modifizierenden Peptid oder Protein Peptidbindungen, die der Spezifität der für die Einführung eingesetzten Serin- oder Cysteinpeptidase entsprechen, dann wird entweder eine andere Peptidase mit der entsprechenden Spezifitätsdeterminante in der Abgangsgruppe des nichtpeptidischen Acyldonors eingesetzt, von der keine sensitiven Peptidbindungen in der Zielsequenz gespalten werden können, oder man führt die biokatalytische Modifizierung im gefrorenen Zustand durch (vgl. Review: M. Hänsler, H.-D. Jakubke, J. Peptide Sei. 2 (1996) 279-289), wobei neben hohen Umsatzraten unerwünschte proteolytische Spaltungen ausgeschlossen werden.If there are peptide bonds in the peptide or protein to be modified which correspond to the specificity of the serine or cysteine peptidase used for the introduction, then either another peptidase with the corresponding specificity determinant is used in the leaving group of the non-peptide acyl donor, of which no sensitive peptide bonds in the Target sequence can be cleaved, or one carries out the biocatalytic modification in the frozen state (cf. Review: M. Hänsler, H.-D. Jakubke, J. Peptide Sci. 2 (1996) 279-289), whereby besides high conversion rates undesirable proteolytic cleavages can be excluded.
Die modifizierten Peptide und Proteine können mit üblichen Methoden der Proteinchemie separiert und gereinigt werden.The modified peptides and proteins can be separated and purified using conventional methods in protein chemistry.
Die vorliegende Erfindung wird im folgenden anhand von Beispielen näher ausgeführt.
Beispiel 1 - V8-Protease-katalysierte N-terminale Einführung von 2-Aminobenzoesäure in PeptideThe present invention is explained in more detail below with the aid of examples. Example 1 - V8 protease-catalyzed N-terminal introduction of 2-aminobenzoic acid into peptides
Für die Modellreaktion wurde als Carboxykomponente 2-Aminobenzoesäure- carboxymethylthioester, im folgenden mit 2-ABz-SCm bezeichnet, und als Aminokomponente das Decapeptid Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly verwendet. 2-ABz-SCm und Aminokomponente wurden in einem Verhältnis von 2 : 1 in einer Konzentration von 4 mM bzw. 2 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil an organischem Lösungsmittel. Die Reaktion wurde durch Zugabe des Enzyms gestartet und nach praktisch vollständigem Umsatz von 2-ABz-SCm durch eine Inaktivierung des Enzyms beendet. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Die Enzymkatalyse führte zu einer 99%igen Umwandlung von Leu-Ala-Leu-Ala-Ser-Ala-Ser- Ala-Phe-Gly in das entsprechend N-terminal 2-ABz-modifizierte Analoga. Die Identität des Syntheseproduktes wurde durch die üblichen Methoden der Organischen Chemie überprüft.For the model reaction, 2-aminobenzoic acid carboxymethylthioester, hereinafter referred to as 2-ABz-SCm, was used as the carboxy component, and the decapeptide Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly was used as the amino component. 2-ABz-SCm and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively. An aqueous buffer system with a small proportion of organic solvent was used as the solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of 2-ABz-SCm, ended by inactivating the enzyme. The analysis and quantification of the reaction was carried out using chromatographic methods. The enzyme catalysis led to a 99% conversion of Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly into the corresponding N-terminal 2-ABz-modified analogues. The identity of the synthesis product was checked by the usual methods of organic chemistry.
Beispiel 2 - V8-Protease-katalysierte N-terminale Einführung der Phloretyl-Gruppe in PeptideExample 2 - V8 protease-catalyzed N-terminal introduction of the phloretyl group into peptides
Für die Modellreaktion wurde als Carboxykomponente Phloretyl-carboxymethylthioester, im folgenden mit Phloretyl-SCm bezeichnet, und als Aminokomponente das Decapeptid Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly verwendet. Phloretyl-SCm und Aminokomponente wurden in einem Verhältnis von 2 : 1 in einer Konzentration von 4 mM bzw. 2 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil an organischem Lösungsmittel. Die Reaktion wurde durch Zugabe des Enzyms gestartet und nach praktisch vollständigem Umsatz von Phloretyl- SCm durch eine Inaktivierung des Enzyms beendet. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Die Enzymkatalyse führte zu einer 99.7%igen Umwandlung von Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly in das entsprechend N-terminal Phloretyl-modifizierte Analoga. Die Identität des Syntheseproduktes wurde durch die üblichen Methoden der Organischen Chemie
überprüft. Die Reaktion führte weder zu einer Λf-Modifizierung des in der Aminokomponente befindlichen Lysins, noch zu einer detektierbaren proteolytischen Spaltung nach Asparaginsäure.For the model reaction, the carboxy component used was phloretyl-carboxymethylthioester, hereinafter referred to as phloretyl-SCm, and the amino component was the decapeptide Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly. Phloretyl-SCm and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively. An aqueous buffer system with a small proportion of organic solvent was used as the solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of phloretyl-SCm, ended by inactivating the enzyme. The analysis and quantification of the reaction was carried out using chromatographic methods. Enzyme catalysis led to a 99.7% conversion of Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly into the corresponding N-terminal phloretyl-modified analogue. The identity of the synthesis product was determined by the usual methods of organic chemistry checked. The reaction did not lead to a Λf modification of the lysine in the amino component, nor to a detectable proteolytic cleavage after aspartic acid.
Beispiel 3 - -Chymotrypsin-katalysierte N-terminale Einführung von 2- Aminobenzoesäure in PeptideExample 3 -Chymotrypsin-Catalyzed N-Terminal Introduction of 2-Aminobenzoic Acid into Peptides
Für die Modellreaktion wurde als Carboxykomponente 2-Aminobenzoesäure-4- guanidinophenylester, im folgenden mit 2-ABz-OGp bezeichnet, und als Aminokomponente das Oligopeptid Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala- Gly-Ala-Tyr verwendet. 2-ABz-OGp und Aminokomponente wurden in einem Verhältnis von 2 : 1 in einer Konzentration von 4 mM bzw. 2 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil an organischem Lösungsmittel. Die Reaktion wurde durch Zugabe des Enzyms gestartet und nach praktisch vollständigem Umsatz von 2-ABz-OGp durch eine Inaktivierung des Enzyms beendet. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Die Enzymkatalyse führte zu einer 98,8%igen Umwandlung von Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr in das entsprechend N-terminal 2-ABz-modifizierte Analoga. Die Identität des Syntheseproduktes wurde durch die üblichen Methoden der Organischen Chemie überprüft. Die Reaktion führte weder zu einer Modifizierung trifunktioneller Seitenketten, noch zu einer detektierbaren proteolytischen Spaltung.For the model reaction, 2-aminobenzoic acid, 4-guanidinophenyl ester was designated as the carboxy component, hereinafter referred to as 2-ABz-OGp, and the oligopeptide Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys as the amino component -Ala-Ala- Gly-Ala-Tyr used. 2-ABz-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively. An aqueous buffer system with a small proportion of organic solvent was used as the solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of 2-ABz-OGp, ended by inactivating the enzyme. The analysis and quantification of the reaction was carried out using chromatographic methods. The enzyme catalysis led to a 98.8% conversion of Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr into the corresponding N-terminal 2 -ABz-modified analogues. The identity of the synthesis product was checked by the usual methods of organic chemistry. The reaction neither led to a modification of trifunctional side chains, nor to a detectable proteolytic cleavage.
Beispiel 4 - α-Chymotrypsin-katalysierte N-terminale Einführung der Phloretyl-Gruppe in PeptideExample 4 - α-Chymotrypsin-Catalyzed N-Terminal Introduction of the Phloretyl Group into Peptides
Für die Modellreaktion wurde als Carboxykomponente Phloretyl-4- guanidinophenylester, im folgenden mit Phloretyl-OGp bezeichnet, und als Aminokomponente das Oligopeptid Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala- Gly-Ala-Tyr verwendet. Phloretyl-OGp und Aminokomponente wurden in einem Verhältnis von 2 : 1 in einer Konzentration von 4 mM bzw. 2 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil an
organischem Lösungsmittel. Die Reaktion wurde durch Zugabe des Enzyms gestartet und nach praktisch vollständigem Umsatz von Phloretyl-OGp durch eine Inaktivierung des Enzyms beendet. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Die Enzymkatalyse führte zu einer 99.3%igen Umwandlung von Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly in das entsprechend N- terminal Phloretyl-modifizierte Analoga. Die Identität des Syntheseproduktes wurde durch die üblichen Methoden der Organischen Chemie überprüft. Die Reaktion führte weder zu einer Modifizierung trifunktioneller Seitenketten, noch zu einer detektierbaren proteolytischen Spaltung.For the model reaction, the carboxy component was phloretyl-4-guanidinophenyl ester, hereinafter referred to as phloretyl-OGp, and the amino component was the oligopeptide Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala - Gly-Ala-Tyr used. Phloretyl-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively. An aqueous buffer system with a small proportion of was used as the solvent organic solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of phloretyl-OGp, ended by inactivating the enzyme. The analysis and quantification of the reaction was carried out using chromatographic methods. Enzyme catalysis led to a 99.3% conversion of Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly into the corresponding N-terminal phloretyl-modified analogues. The identity of the synthesis product was checked by the usual methods of organic chemistry. The reaction neither led to a modification of trifunctional side chains, nor to a detectable proteolytic cleavage.
Beispiel 5 - Trypsin-katalysierte N-terminale Einführung von 2-Aminobenzoesäure in PeptideExample 5 - Trypsin-catalyzed N-terminal introduction of 2-aminobenzoic acid into peptides
Für die Modellreaktion wurde als Carboxykomponente 2-Aminobenzoesäure-4- guanidinophenylester, im folgenden mit 2-ABz-OGp bezeichnet, und als Aminokomponente das Decapeptid Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly verwendet. 2-ABz-OGp und Aminokomponente wurden in einem Verhältnis von 2 : 1 in einer Konzentration von 4 mM bzw. 2 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil an organischem Lösungsmittel. Die Reaktion wurde durch Zugabe des Enzyms gestartet und nach praktisch vollständigem Umsatz von 2-ABz-OGp durch eine Inaktivierung des Enzyms beendet. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Die Enzymkatalyse führte zu einer 94,4%igen Umwandlung von Leu-Ala-Leu-Ala-Ser-Ala- Ser-Ala-Phe-Gly in das entsprechend N-terminal 2-ABz-modifizierte Analoga. Die Identität des Syntheseproduktes wurde durch die üblichen Methoden der Organischen Chemie überprüft.For the model reaction, 2-aminobenzoic acid-4-guanidinophenyl ester, hereinafter referred to as 2-ABz-OGp, was used as the carboxy component, and the decapeptide Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly was used as the amino component. 2-ABz-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively. An aqueous buffer system with a small proportion of organic solvent was used as the solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of 2-ABz-OGp, ended by inactivating the enzyme. The analysis and quantification of the reaction was carried out using chromatographic methods. The enzyme catalysis led to a 94.4% conversion of Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly into the corresponding N-terminal 2-ABz-modified analogues. The identity of the synthesis product was checked by the usual methods of organic chemistry.
Beispiel 6 - Trypsin-katalysierte N-terminale Einführung der Phloretyl-Gruppe in PeptideExample 6 - Trypsin-catalyzed N-terminal introduction of the phloretyl group into peptides
Für die Modellreaktion wurde als Carboxykomponente Phloretyl-4- guanidinophenylester, im folgenden mit Phloretyl-OGp bezeichnet, und als Aminokomponente das Decapeptid Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly
verwendet. Phloretyl-OGp und Aminokomponente wurden in einem Verhältnis von 2 : 1 in einer Konzentration von 4 mM bzw. 2 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil an organischem Lösungsmittel. Die Reaktion wurde durch Zugabe des Enzyms gestartet und nach praktisch vollständigem Umsatz von Phloretyl-OGp durch eine Inaktivierung des Enzyms beendet. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Die Enzymkatalyse führte zu einer quantitativen Umwandlung von Leu-Ala-Leu-Ala-Lys-Ala- Asp-Ala-Phe-Gly in das entsprechend N-terminal Phloretyl-modifizierte Analoga. Die Identität des Syntheseproduktes wurde durch die üblichen Methoden der Organischen Chemie überprüft.For the model reaction, the carboxy component was phloretyl-4-guanidinophenyl ester, hereinafter referred to as phloretyl-OGp, and the amino component was the decapeptide Leu-Ala-Leu-Ala-Ser-Ala-Ser-Ala-Phe-Gly used. Phloretyl-OGp and amino component were used in a ratio of 2: 1 in a concentration of 4 mM and 2 mM, respectively. An aqueous buffer system with a small proportion of organic solvent was used as the solvent. The reaction was started by adding the enzyme and, after virtually complete conversion of phloretyl-OGp, ended by inactivating the enzyme. The analysis and quantification of the reaction was carried out using chromatographic methods. Enzyme catalysis led to a quantitative conversion of Leu-Ala-Leu-Ala-Lys-Ala-Asp-Ala-Phe-Gly into the corresponding N-terminal phloretyl-modified analogue. The identity of the synthesis product was checked by the usual methods of organic chemistry.
Beispiel 7 - Biotinylierung von E. coli Parvulin 10Example 7 - Biotinylation of E. coli Parvulin 10
Für die Modellreaktion wurde als Carboxylkomponente BiotinyI-4-Guanidinophenylester, im folgenden mit Biotinyl-OGp bezeichnet und als Aminokomponente das Protein E. coli Parvulin 10 verwendet. Biotinyl-OGp und Parvulin wurden in einem Verhältnis von 1 :4 in einer Konzentration von 2mM bzw. 8mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem mit einem geringen Anteil eines organischen Lösungsmittels. Konkret wurde 0,1 M HEPES (N2-[2-Hydroxyethyl]Piperazin-N'-[2-Ethansulfonsäure) Puffer pH 8,0, 0,1 M NaCI, 0,01 M CaCI2 und 8 % (v/v) DMF (Dimethylformamid) verwendet. Die Reaktion wurde durch Zugabe des Enzyms Trypsin D189K + K60E (Trypsinmutante, bei der die Aminosäure D an Position 189 durch K bzw. K an Position 60 durch E ersetzt ist; Herstellung siehe Beispiel 9) gestartet und nach 2 Stunden Reaktionszeit beendet. Das Enzym wurde in einer Konzentration von 6,5 x 10"6 M eingesetzt. Die Analyse und Quantifizierung der Reaktion erfolgte durch MALDI-MS Spektroskopie (Figur 1). Die Enzymkatalyse führte zu einer Umwandlung von E. coli Parvulin 10 in das N-terminal biotinylierte Biotinyl-(E. coli Parvulin 10).For the model reaction, the carboxyl component was BiotinyI-4-guanidinophenyl ester, hereinafter referred to as Biotinyl-OGp, and the protein E. coli Parvulin 10 was used as the amino component. Biotinyl-OGp and parvulin were used in a ratio of 1: 4 in a concentration of 2mM and 8mM, respectively. An aqueous buffer system with a small proportion of an organic solvent was used as the solvent. Specifically, 0.1 M HEPES (N 2 - [2-hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid) buffer pH 8.0, 0.1 M NaCl, 0.01 M CaCl 2 and 8% (v / v) DMF (dimethylformamide) used. The reaction was started by adding the enzyme trypsin D189K + K60E (trypsin mutant in which the amino acid D at position 189 has been replaced by K or K at position 60 by E; preparation see Example 9) and ended after a reaction time of 2 hours. The enzyme was used in a concentration of 6.5 x 10 "6 M. The analysis and quantification of the reaction was carried out by MALDI-MS spectroscopy (FIG. 1). The enzyme catalysis led to a conversion of E. coli parvulin 10 into the N- terminally biotinylated biotinyl (E. coli parvulin 10).
Die Primärsequenz von E. coli Parvulin 10 ist bekannt und entspricht der folgenden Aminosäure-Sequenz.
AKTAAALHIL VKEEKLALDL LEQIKNGADF GKLAKKHSIC PSGKRGGDLG EFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RNThe primary sequence of E. coli parvulin 10 is known and corresponds to the following amino acid sequence. AKTAAALHIL VKEEKLALDL LEQIKNGADF GKLAKKHSIC PSGKRGGDLG EFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RN
Beispiel 8 - Biotinylierung von RNase T1Example 8 - Biotinylation of RNase T1
Für die Modellreaktion wurde als Carboxylkomponente Biotinyl-OGp und als Aminokomponente das Protein RNase T1 verwendet. Für die Biotinylierung von RNase T1 wurde eine Variante mit zusätzlichem Arg(R)-Gly(G)-Rest am N-Terminus des Proteins eingesetzt. Durch unterschiedliche in vivo Prozessierung des Vorläuferproteins wurde jedoch neben der gewünschten RNase T1 -Variante (RG-RNase T1) auch die um eine Aminosäure verkürzte Spezies (G-RNase T1) als auch der Wildtyp (RNase T1) erhalten. Dieses Gemisch wurde ohne Auftrennung der einzelnen Varianten für die Enzym-katalysierte Biotinylierung eingesetzt. Biotinyl-OGp und RNase T1 -Gemisch wurden in einem Verhältnis von 1 :4 in einer Konzentration von 2 mM bzw. 8 mM eingesetzt. Als Lösungsmittel diente ein wässriges Puffersystem wie in Beispiel 7 beschrieben. Die Reaktion wurde duch Zusatz des Enzyms Trypsin D189K + K60E (6,5 x 10"6 M) gestartet. Die Reaktionszeit betrug 2 Stunden. Die Analyse und Quantifizierung der Reaktion erfolgte durch chromatographische Methoden. Das Elektropherogramm der durchgeführten Kapillarelektrophorese ist in Figur 2 dargestellt. Es ist deutlich zu erkennen, dass die RG-RNase T1 nahezu quantitativ in die Biotinyl- RG-RNase T1 überführt wurde.For the model reaction, the protein RNase T1 was used as the carboxyl component biotinyl-OGp and as the amino component. A variant with an additional Arg (R) -Gly (G) residue at the N-terminus of the protein was used for the biotinylation of RNase T1. Through different in vivo processing of the precursor protein, in addition to the desired RNase T1 variant (RG-RNase T1), the amino acid-shortened species (G-RNase T1) and the wild type (RNase T1) were also obtained. This mixture was used without separation of the individual variants for the enzyme-catalyzed biotinylation. Biotinyl-OGp and RNase T1 mixture were used in a ratio of 1: 4 in a concentration of 2 mM and 8 mM, respectively. An aqueous buffer system as described in Example 7 was used as the solvent. The reaction was started by adding the enzyme trypsin D189K + K60E (6.5 x 10 "6 M). The reaction time was 2 hours. The analysis and quantification of the reaction were carried out by chromatographic methods. The electropherogram of the capillary electrophoresis carried out is shown in FIG It can be clearly seen that the RG-RNase T1 was converted almost quantitatively into the Biotinyl-RG-RNase T1.
Beispiel 9 - Herstellung der Trypsinmutante D189K+K60EExample 9 - Preparation of the trypsin mutant D189K + K60E
Plasmideplasmids
Zur Durchführung der ortsgerichteten Mutagenesen kam der E. coli Vector pST zum Einsatz. Dieser enthält einen Teil des Bluescript-Vectors sowie das Gen für anionisches Rattentrypsin, das mit einem α-factor-leader sowie einem ADH/GAPDH-Promotor fusioniert ist.
Die Proteinexpression erfolgte mit Hilfe des pYT-Plasmides, einem pBS24-Abkömmling, das Selektions-Marker für Uracil- und Leucin-defizientes Medium trägt.The E. coli Vector pST was used to carry out the site-directed mutagenesis. This contains part of the Bluescript vector and the gene for anionic rat trypsin, which is fused to an α-factor leader and an ADH / GAPDH promoter. Protein expression was carried out using the pYT plasmid, a pBS24 derivative which carries selection markers for uracil- and leucine-deficient medium.
Sowohl das pST- als auch das pYT- Plasmid verfügt über ein Ampicillinresistenz- Gen. Die Karten beider Vektoren, d.h. der Plasmide pST (5,4 kb)- und pYT (14 kb), mit den entsprechenden Schnittstellen sind in Figur 3 dargestellt.Both the pST and pYT plasmids have an ampicillin resistance gene. The maps of both vectors, i.e. the plasmids pST (5.4 kb) and pYT (14 kb), with the corresponding interfaces, are shown in FIG. 3.
MutaqeneseMutagenesis
Die ortsgerichteten Mutagenesen wurden unter Verwendung des Quik change ® -Kit (STRATAGENE) im E. coli Plasmid pST durchgeführt.The site-directed mutagenesis was carried out using the Quik change ® kit (STRATAGENE) in the E. coli plasmid pST.
Das verwendete Verfahren ähnelt einer PCR, wobei ausgehend von zwei synthetischen Oligonukleotidprimern, welche die gewünschte Mutation enthalten, beide Plasmidstränge des pST-Vectors von PFU-Polymerase repliziert werden. Wildtyp pST diente als Template zur Erzeugung von einzelnen Mutationen. Diese Einzelmutanten waren wiederum Ausgangspunkt für die Konstruktion der Doppel-Mutante.The method used is similar to a PCR, with two plasmid strands of the pST vector of PFU polymerase being replicated starting from two synthetic oligonucleotide primers which contain the desired mutation. Wild-type pST served as a template for generating single mutations. These single mutants were the starting point for the construction of the double mutant.
Zum Einsatz kamen folgende Oligonukleotid-Primer, wobei die fettgedruckten Buchstaben die Mutationen angeben:The following oligonucleotide primers were used, the bold letters indicating the mutations:
D189K aa)) 5 5'' - GGA GGC AAG AAC GAT TCC TGC - 3' b) 5 ' - GCA GGA ATC GTT CTT GCC TCC - 3' K60E aa)) 5 5'' - CAC TGC TAT GAG TCC CGC ATC - 3' b) 5 ' - GAT GCG GGA CTG ATA GCA GTG - 3'D189K aa)) 5 5 '' - GGA GGC AAG AAC GAT TCC TGC - 3 'b) 5' - GCA GGA ATC GTT CTT GCC TCC - 3 'K60E aa)) 5 5' '- CAC TGC TAT GAG TCC CGC ATC - 3 'b) 5' - GAT GCG GGA CTG ATA GCA GTG - 3 '
Das erhaltene PCR-Produkt wurde in ultrakompetente E. coli XL II blue Zellen (STRATAGENE) transformiert. Die anschließende Selektion erfolgte auf Ampicillin- haltigen Nähragarplatten (LB-amp). Die gepickten Kolonien wurden in ein Ampicillin- haltiges Flüssigmedium (LB-amp) überführt wobei nach eintägiger Kultivierung die Isolierung des Plasmides unter Verwendung des SNAP-Kit (INVITROGENE) durchgeführt wurde. Die Kontrolle der isolierten DNA erfolgte mittels Elektrophorese mit einem 1%-
igen Agarose-Gel. Durch Sequenzierung des kompletten Gens konnte sichergestellt werden, dass nur die gewünschten Mutationen enthalten waren.The PCR product obtained was transformed into ultra-competent E. coli XL II blue cells (STRATAGENE). The subsequent selection was carried out on nutrient agar plates (LB-amp) containing ampicillin. The picked colonies were transferred to a liquid medium containing ampicillin (LB-amp) and, after culturing for one day, the plasmid was isolated using the SNAP kit (INVITROGENE). The isolated DNA was checked by electrophoresis with a 1% agarose gel. By sequencing the complete gene it was possible to ensure that only the desired mutations were contained.
Subklonierungsubcloning
Für alle Mutanten die im pST-Plasmid erzeugt wurden, war eine Subklonierung im pYT- Expressions-Vektor notwendig. Diese erfolgte durch Restriktionsverdau mit Barn Hl und Sal I und Ligation in das korrespondierende pYT-Vektorfragment. Alle Vektorfragmente wurden im entsprechenden Restriktionsmix auf ein niedrig schmelzendes Agarose-Gel (0,8%) aufgetragen und nach ausreichender Auftrennung ausgeschnitten. Die Gelstücke wurden bei 55 °C geschmolzen und nach gewünschter Kombination vereinigt und bei 16 °C über Nacht mit T4 DNA-Ligase ligiert. Die abermals notwendige Transformation und Plasmidisolierung erfolgte wie oben beschrieben.Subcloning in the pYT expression vector was necessary for all mutants generated in the pST plasmid. This was done by restriction digestion with Barn HI and Sal I and ligation into the corresponding pYT vector fragment. All vector fragments were applied in the appropriate restriction mix to a low-melting agarose gel (0.8%) and cut out after sufficient separation. The gel pieces were melted at 55 ° C. and combined according to the desired combination and ligated at 16 ° C. overnight with T4 DNA ligase. The transformation and plasmid isolation again required was carried out as described above.
Eine erfolgreiche Subklonierung ließ sich durch ein charakteristisches Restriktionsmuster nach Doppelverdau mit Eco Rl und Barn HI im Agarose-Gel nachweisen.Successful subcloning was demonstrated by a characteristic restriction pattern after double digestion with Eco Rl and Barn HI in the agarose gel.
Durch Sequenzierung des kompletten Trypsinogen-Gens konnte sichergestellt werden, dass nur die gewünschten Mutationen enthalten waren.By sequencing the complete trypsinogen gene it was possible to ensure that only the desired mutations were contained.
Hefetranformation und SelektionYeast transformation and selection
Der verwendete Hefezellstamm trägt die Bezeichnung Saccharomyces cerevisiae DLM 101 α [Mat a,leu 2-3,-112 his 2, 3-11,-15 can 1, ura 3Δ, pep4Δ, [cir°], DM 23]. Für die Herstellung kompetenter Hefezellen und die Transformation der pYT- Plasmide kam der EZ-Hefetransformationskit (ZYMO-RESEARCH) zum Einsatz. Die Selektion erfolgte auf Uracil-defizienten SC-Platten durch Bebrütung bei 30 °C für 3 bis 4 Tage. Einzelkolonien wurden weiterüberimpft auf Leucin-defiziente SC-Platten und ebenfalls 3 bis 4 Tage bei 30 °C inkubiert, wodurch die Kopienanzahl des Plasmides in den Zellen zunahm. Einzelkolonien dieser Platten wurden zum Animpfen der Vorkulturen des Leucin-defizienten SC-Flüssigmediums mit 8% Glukose herangezogen. Die Inkubation
erfolgte unter schütteln bei 30 °C und 120 rpm für 3 Tage. Als Inokulum zum Animpfen der 1 Liter Hauptkulturen mit YPD- Medium (1% Glucose, 1% Bactopepton, 0,5% Hefeextrakt) wurden 20 ml Vorkultur eingesetzt. Die Inkubationsparameter entsprachen denen der Vorkultur, wobei nach 4 Tagen geerntet wurde.The yeast cell strain used is called Saccharomyces cerevisiae DLM 101 α [Mat a, leu 2-3, -112 to 2, 3-11, -15 can 1, ura 3Δ, pep4Δ, [cir °], DM 23]. The EZ yeast transformation kit (ZYMO-RESEARCH) was used to produce competent yeast cells and transform the pYT plasmids. The selection was made on uracil-deficient SC plates by incubation at 30 ° C. for 3 to 4 days. Single colonies were further vaccinated on leucine-deficient SC plates and also incubated for 3 to 4 days at 30 ° C, which increased the number of copies of the plasmid in the cells. Individual colonies of these plates were used to inoculate the pre-cultures of the leucine-deficient SC liquid medium with 8% glucose. The incubation was done with shaking at 30 ° C and 120 rpm for 3 days. 20 ml of preculture were used as inoculum for inoculating the 1 liter main cultures with YPD medium (1% glucose, 1% bactopeptone, 0.5% yeast extract). The incubation parameters corresponded to those of the preculture, with harvesting after 4 days.
Isolierung und Reinigung der TrvpsinvariantenIsolation and cleaning of the trinpsin variants
Durch Zentrifugation für 20 min bei 4000 rpm wurden zunächst die Zellen separiert und der auf pH 4,0 eingestellte Überstand erneut bei 12.000 rpm zentrifugiert. Der praktisch partikelfreie Trypsinogen-haltige Überstand wurde auf eine mit 2 mM Natriumacetat/100 mM Essigsäure (pH 4,5) equilibrierte Toyopearl 650 M (SUPELCO) Kationenaustauschersäule aufgetragen. Eluiert wurde mittels eines linearen pH- Gradienten beginnend von 2 mM Natriumacetat/ 100 mM Essigsäure (pH 4,5) bis 200 mM Tris/HCI (pH 8,0).The cells were first separated by centrifugation for 20 min at 4000 rpm and the supernatant adjusted to pH 4.0 was centrifuged again at 12,000 rpm. The practically particle-free trypsinogen-containing supernatant was applied to a Toyopearl 650 M (SUPELCO) cation exchange column equilibrated with 2 mM sodium acetate / 100 mM acetic acid (pH 4.5). Elution was carried out using a linear pH gradient starting from 2 mM sodium acetate / 100 mM acetic acid (pH 4.5) to 200 mM Tris / HCl (pH 8.0).
Durch SDS-Polyacrylamid-Gelelektrophorese unter Verwendung eines 15%-igen Polyacrylamid-Geles konnten die Trypsinogen enthaltenden Fraktionen ermittelt und zusammengefasst werden. Das Volumen der Proteinlösungen wurde mit Hilfe von Centriprep-Konzentratoren (AMICON) auf etwa 10 bis 15 ml eingeengt.The trypsinogen-containing fractions could be determined and summarized by SDS-polyacrylamide gel electrophoresis using a 15% polyacrylamide gel. The volume of the protein solutions was reduced to about 10 to 15 ml using Centriprep concentrators (AMICON).
Die Aktivierung der Trypsinogen-Variante zum entsprechenden Trypsin D189K+K60E erfolgte mittels hochaufgereinigter Enterokinase (BIOZYME) bei pH 6,5 und wurde durch SDS-Gelelktrophorese kontrolliert.The activation of the trypsinogen variant to the corresponding trypsin D189K + K60E was carried out using highly purified enterokinase (BIOZYME) at pH 6.5 and was monitored by SDS gel electrophoresis.
Unter Verwendung eines Biocad Sprint Perfusionschrommatographie-Systems (PERSEPTIVE BIOSYSTEMS) wurde die Aufreinigung des aktivierten Enzyms durchgeführt. Die Auftrennung der Proteinproben erfolgte auf einer mit 5%-igen Bis-/Tris-Propan pH 6,0 equilibrierten PORÖS 20 HQ - Anionenaustauscher-Säule (4 x 100 mm, PERSEPTIVE BIOSYSTEMS) und nachfolgender Gradientenelution bis 95% 3M NaCI- Lösung. Die Trypsin-enthaltenden Fraktionen wurden mit Hilfe eines SDS-Geles auf Reinheit überprüft und zusammengefasst. Abschließend erfolgte die Dialyse gegen 1
mM HCI bei 4°C und Einengung der Proben mit Centriprep-Konzentratoren auf 2 bis 4 ml.The activated enzyme was purified using a Biocad Sprint Perfusion Chromatography System (PERSEPTIVE BIOSYSTEMS). The protein samples were separated on a PORÖS 20 HQ - anion exchange column (4 x 100 mm, PERSEPTIVE BIOSYSTEMS) equilibrated with 5% bis- / tris-propane pH 6.0 and subsequent gradient elution up to 95% 3M NaCI solution. The fractions containing trypsin were checked for purity using an SDS gel and pooled. Finally, dialysis was carried out against 1 mM HCI at 4 ° C and concentration of the samples with Centriprep concentrators to 2 to 4 ml.
Die Endausbeuten beliefen sich auf etwa 2 bis 5 mg Protein pro Liter Kulturmedium.The final yields were about 2 to 5 mg protein per liter of culture medium.
Konzentrationsbestimmungconcentration determination
Die Proteinkonzentration der Präparate wurde nach der Methode von Bradford an einem Spektrophotometer bei einer Wellenlänge von 595 nm bestimmt. Die Aufnahme der Eichkurve erfolgte anhand einer Rindertrypsin-Verdünnungsreihe zwischen 50 μm/ml und 1 mg/ml.
The protein concentration of the preparations was determined using the Bradford method on a spectrophotometer at a wavelength of 595 nm. The calibration curve was recorded using a series of bovine trypsin dilutions between 50 μm / ml and 1 mg / ml.
Claims
1. Verfahren zur biokatalytischen Modifizierung von Peptiden und Proteinen dadurch gekennzeichnet, dass als Biokatalysator eine Peptidase in Verbindung mit einem nichtaminosäure- oder nichtpeptidartigen Substratmimetikum eingesetzt wird.1. Process for the biocatalytic modification of peptides and proteins, characterized in that a peptidase is used as the biocatalyst in conjunction with a non-amino acid or non-peptide-like substrate mimetic.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass als Acylierungskomponente ein Carbonsäureester verwendet wird, dessen Acylteil der Modifizierungsgruppe entspricht und dessen Abgangsgruppe die Spezifitätsdeterminante der für die Katalyse eingesetzten Peptidase enthält.2. The method according to claim 1, characterized in that a carboxylic acid ester is used as the acylation component, the acyl part of which corresponds to the modification group and the leaving group contains the specificity determinant of the peptidase used for the catalysis.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die Reaktion in einem wäßrigen Medium bei Raumtemperatur oder in einem gefrorenen wäßrigen System oder bei tiefen Temperaturen zwischen - 5 und - 20° C durchgeführt wird.3. The method according to claim 1 or 2, characterized in that the reaction is carried out in an aqueous medium at room temperature or in a frozen aqueous system or at low temperatures between - 5 and - 20 ° C.
4. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass als Peptidasen Trypsin, Chymotrypsin, V8 Protease, Glu-spezifische Endopeptidase aus Bacillus licheniformis, Subtilisin u.a. bzw. Mutanten dieser Enzyme oder Enzyme mit ähnlichen Spezifitätsdeterminanten eingesetzt werden.4. The method according to any one of the preceding claims, characterized in that as peptidases trypsin, chymotrypsin, V8 protease, Glu-specific endopeptidase from Bacillus licheniformis, subtilisin and others. or mutants of these enzymes or enzymes with similar specificity determinants.
5. Verwendung von Peptidasen, dadurch gekennzeichnet, dass sie zum biokatalytischen Einführen von Marker- und Reportergruppen in Peptide und Proteine eingesetzt werden. 5. Use of peptidases, characterized in that they are used for the biocatalytic introduction of marker and reporter groups into peptides and proteins.
6. Verwendung von Peptidasen nach Anspruch 5, dadurch gekennzeichnet, dass sie zum biokatalytischen Einführen von Marker- und Reportergruppen in Peptide und Proteine unter Vermeiden derer reversiblen enzymatischen Abspaltung eingesetzt werden, indem die einzuführende Marker - oder Reportergruppe ein Esterderivat als Abgangsgruppe trägt, welche die nativeSpezifität des eingesetzten Enzyms ausschaltet.6. Use of peptidases according to claim 5, characterized in that they are used for the biocatalytic introduction of marker and reporter groups into peptides and proteins while avoiding their reversible enzymatic cleavage by the marker or reporter group to be introduced carrying an ester derivative as the leaving group switches off the native specificity of the enzyme used.
7. Verwendung von Peptidasen nach Anspruch 5 und 6, dadurch gekennzeichnet, dass als Peptidasen Trypsin, Chymotrypsin, V8 Protease, Glu-spezifische Endopeptidase aus Bacillus licheniformis, Subtilisin oder Mutanten dieser Enzyme oder Enzyme mit ähnlichen Spezifitätsdeterminanten eingesetzt werden. 7. Use of peptidases according to claims 5 and 6, characterized in that trypsin, chymotrypsin, V8 protease, Glu-specific endopeptidase from Bacillus licheniformis, subtilisin or mutants of these enzymes or enzymes with similar specificity determinants are used as peptidases.
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US20110045530A1 (en) * | 2008-02-13 | 2011-02-24 | Peter Jan Leonard Mario Quaedflieg | Enzymatic conjugation of bioactive moieties |
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DK67691D0 (en) * | 1991-03-01 | 1991-04-15 | Carlbiotech Ltd As | ENZYMATIC PROCEDURE FOR C-TERMINAL MODIFICATION OF PEPTIDES AND INTERMEDIATES FOR USE BY THE PROCEDURE |
US5985627A (en) * | 1997-02-28 | 1999-11-16 | Carlsberg Laboratory | Modified carboxypeptidase |
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