IL94526A - Method for c-terminal modification of proteins - Google Patents

Description

Method for C-tennlnal modification of proteins FORSCHUNGSZENTRUM JULICH GmbH C. 80577 - 1 94526/2 The present invention relates to a method for the C-terminal modification of proteins with at least one chemically reactive or recognition specific modifier which is linked to the protein by peptidation via a L-amino acid or a L-amino acid derivative as coupling member under the formation of the amide.

Methods for the modification of proteins with certain functional groups such as marker groups, immobilisation groups or activation groups are known in which modifications via lysine groups in the protein is achieved more, or less by accident. Other groups in proteins such as tyrosine or cystein are also non-specifically modified by these methods.

The known methods result scarcely in specifically modified products and the activity of the modified protein is usually considerably decreased.

K. Rose et al. (Europ. Peptid-Symp. Tiibigen, BRD, September, 1988, Poster No. 64) discloses a principle for C-terminal modification of proteins which add to the C- terminal a coupling member with the aid of proteolytic enzymes to which a modifier is linked in ¾ second step .

C-terminal modification of proteins is already reported by K. Hofmann et al. (J. Am. Chem. Soc. 1978, page 3585 ff) however, this was achieved by chemical reactions.

The above methods for C-terminal modification of proteins are relatively complicated.

Therefore it is an object of the invention to provide a method with which C-terminal modification of a protein can be achieved in a relatively simple manner.

This object is attained in that the protein is enzymatically transformed into a protein which is modified once or more at the C-terminal, the reaction being carried out with an amino acid or amino acid derivative having a free a-amino group and carrying at least one modifier and in the presence of protease.

Although enzymatic C-terminal coupling of amino acids or amino acid amides or esters to peptides in the presence of protease, especially carboxypeptidase Y (CPD-Y), has been known for some time (see U.S. Patent 4,339,534), this enzymatic reaction has not been used hitherto for C-terminal linking of chemically reactive modifiers or complicated recognition specific connection groups.

Proteolytic enzymes such as trypsin or CPD-Y have hitherto only been employed for C-terminal trans-peptization (K. Morihara et al., Biochem. J. 240 (1986), 803; Nature 280 (1979) 412 as well as K. Breddam et al., Carlsberg Res. Commun. 46 (1981) 121) as well as for the synthesis of polypeptides (i.e. K. Rose et al., Biochem. J. 249 (1988) 83-88).

Coupling of chemically reactive or relatively complicated modifiers to proteins by simple enzymatic reaction, which, according to R.E. Feeny, Int. J. Peptide Protein Res. 29, 1987, 145-161, is being developed vigorously since 40 years, has so far not been achieved.

According to the invention (at least) one modifier is coupled intentionally to the C-terrainal of the protein such that it is changed unequivocally by a marking functional group, an immobilisation functional group, an activation functional group or the like. As coupling member serves an at least trifunctional amino acid derivative, the third (or higher) functional group of which may be formed by an amino, thiol, hydroxy, carboxy or similar group. Examples for amino acids with a third functional group which may be employed as coupling members are especially lysine, cystein, glutamic acid, aspartic acid etc.

For the enzymatic C-terminal protein modification according to the invention also a simple amino acid may be employed, the modifier being coupled via its carboxy functional group. Analogously a trifunctional amino acid may serve for the modification which has one or two (identical or similar) modifier(s) coupled via a third functional group and/or the carboxy functional group. If the carboxy functional group is not occupied, an acid derivative, especially an acid amide is employed for the enzymatic reaction.

Special coupling members with modifiers X for a single modification correspond especially to the following formulae X - NH - (CH2)n - CH - CO H2 ( I ) I H2 with n = 1 - 10 or X - CO - (CH2)n - CH - CONH2 (ID H2 with n = 1 - 10 or (CH2)n - CH - CO H2 (III) H2 with n = 1 - 10.

In the compounds of the above formulae (I) to (III) as well as with compounds of formula (V) the modifiers X may be distanced from the amino functional group which was coupled enzymatically to the protein by a further spacer inserted in front of the coupling functional group, for example with formation of (CH2)m - CO - NH - (CH2)n - CH - C0NH2 (IV) with m = 1 bis 10.

In these (possibly extended) coupling members of the above formulae (I) to (III) the amide radicals may be replaced by a further modifier or may contain such a modifier. The compounds to be reacted with the proteins will then correspond, for example, to the formula X - NH - (CH2)n - CH - CO - ( H)p - X' (V) NH2 wherein modifiers X and X' may be identical or different, p = 1 or 0 and n has the abovementioned meaning.

A modifier coupled via the carboxy functional group may also be connected to the protein via a simple L-amino acid C-terminal by employing amino acids of the general formula NH<- As modifiers X or X', for example, biotin, alkanoic acid maleic imide, pyridyldisulfide, phenylazide, etc. may be coupled under formation of an amide, an ester or a thioester or the like.

Depending on the field of application the following modifiers may be distinguished: - as marking groups, for example biotin - as fluorescence markers, for example dansyl-, dabsyl-, fluororescin. - as wetting agents, for example maleic imide-, 2-pyridyl-disulfide, (4-iodoacetyl)-aminobenzoate - as photoreactive wetting agents, for example 4-azidosalicylic acid, 2-(p-azidosalicylic amido)-ethyl-1,3' -dithiopropionate.

The reaction is effected in aqueous organic solution whereby as organic compound polar solvents, especially dimethylsulfoxide or dimethylformamide may be employed. Protein to protease ratios of from 10:1 to 100:1 are especially suitable and the nucleophile to be coupled is employed in large excess. The pH value should be in the range of from 7 to 10, especially at about 8.5. The reaction temperature should be as low as possible and should not exceed room temperature, the reaction is advantageously carried out under cooling of the solution, preferably at about 4"C. The reaction time amounts to several hours. The reaction mixture obtained is worked up by chromatography, gel filtration and possibly by concentration of the modified protein obtained.

In order to prove the usefulness of the method according to the invention there were prepared in particular proteins which were C-terminal modified by biotin or maleic-imidoprOpionyl.

EXAMPLE 1 Preparation of C-terminal biotinylated proteins ε-Biotinyl-L-lysin-amide (biocytinamide) prepared according to modified instructions by Hoffmann et al., J. Am. Chem. Soc. 100 (1978) 3585, was dissolved in a concentration of 100 mg/ml in a mixture of water, DMF and ethanol (2:1:1). The pH was adjusted to 8.5 with 5 M NaOH. The protein to be modified and CPD-Y were added to this solution in a molar ratio of 100:1 or 50:1 and the reaction was carried out overnight at a temperature of 4°C.

The reaction mixture was then separated on a Sephade £yG-25 fine column with 1 M acetic acid as eluent and the protein fraction was collected.

The solution may then be concentrated by lyophilisation or ultrafiltration.

CHARACTERIZATION The incorporation of biotin into the protein could be shown by dot blots stained with alkali phosphatase complex with avidin (M. Wilchek et al., Biochem. Biophys. Res. Comraun., 138 (1986) 872). The C-terminal biotinylated protein is characterized by a single band in native PAGE and blot transfer on a nitrocellulose membrane; it migrates to the same spot as the corresponding chemically biotinylated protein. This shows that the proteins are not further hydrolysed under these reaction conditions.

The following proteins may "be biotinylated successfully: insulin, trypsin, myoglobin, cytochrome-C, R Ase, lysozyme, BSA and human albumin.

EXAMPLE 2 C-terminal ia1el i idopropionylation of proteins 1 g of maleic imidopropionyl hydroxy succinimide ester was dissolved in dry DMF and the solution reacted with 2.4 g of a-B0C-L-Lys-NH2· The reaction mixture was stirred for 24 hours at room temperature. Addition of dry ether precipitated a-BOC-ε-maleic imidopropionyl-L-Lys-NHj . The precipitate was filtered off, washed with dry ether and the a-BOC protecting group was separated with 4 N HC1 in dioxane.

The obtained maleic imidopropionyl lysinamide was reacted enzymatically in the same manner as the biocytinamide according to Example 1 with proteins to obtain the corresponding C-terminal modified proteins.

CHARACTERIZATION The modified proteins (insulin, myoglobin, Lysozyme and cytochrome-C) and the corresponding non-modified proteins were treated with mercaptoethanol. The excess of mercaptoethanol was back-titrated with Ellmann's Reagent. For comparison purposes non-modified proteins were also treated with mercaptoethanol and the back-titration was effected in the same manner. All modified proteins showed less staining than the non-modified proteins.

The degree of modification was estimated by immobilisation on SH-modified Eupergit ®. For this purpose 40 mg SH-modified Eupergit ® was shaken with 1 ml of the modified protein (c = 0.25 mg/ml) at room temperature and pH of 7.8 for 1 hour. After centrifuging the supernatant was collected and the globes treated with mercaptoethanol. After renewed centrifuging the optical density of the supernatant is measured before immobilising and after- immobilising. The estimated degrees of modification were shown to be between 75% for cytochrome-C and 40% for insulin.

According to the invention C-terminal modified proteins may be coupled to each other without difficulty: The coupling of two proteins via their C-terminals was achieved by addition of DTT to a solution of modified myoglobin at pH 7.5 and shaking during one hour. The doubling of the molecular weight from about 17,000 to about 34,000 was demonstrated by native PAGE with corresponding molecular markers.

Claims (8)

- 9 - 94526/3 CLAIMS :

1. A method for the C-terminal modification of proteins with at least one chemically reactive or recognition specific modifier which is linked to the protein by peptidation via a L-amino acid or a L-amino acid derivative as coupling member under the formation of the amide, characterized in that the protein is enzymatically transformed into a protein which is modified once or more at the C-terminal, the reaction being carried out with an amino acid or amino acid derivative having a free a-amino group and carrying at least one modifier and in the presence of protease.

2. A method according to claim 1, characterized in that the modifier is attached to the amino acid via an inserted inert spacer, especially via an -(CH2)m -chain wherein m = 1 - 10, the inserted spacer being on each side connected by a suitable joining group.

3. A method according to Claims 1 or 2, characterized in that carboxypeptidase Y is employed as the protease.

4. A method according to Claims 1-3, characterized in that a trifunctional L-amino acid derivative is employed which has a modifier or modifiers coupled via the third functional group and/or the carboxy functional group.

5. A method according to Claim 4, characterized in that a trifunctional L-amino acid derivative is employed which has two different modifiers coupled via the third functional group and the carboxy group.

6. A method according to Claim 5, characterized in that a trifunctional L-amino amide is employed which has a modifier coupled via its third functional group.

7. A method according to Claim 6, characterized in that the protein is reacted with an G-N-modified L-lysine amide.

8. A method according to any one of the preceding claims substantially as described herein with reference to the Examples. For the Applicants, DR. REINHOLD COHN AND PARTNERS 80577spc.JP/le/18.l.199S