The present invention relates to the cleavage of proteins and peptides, especially the cleavage of fusion proteins and peptides obtained in recombinant DNA methods. When producing peptides and proteins in bacteria by recombinant DNA techniques, it is often necessary to produce the peptide or protein fused to a bacterial protein in order to protect the protein or peptide against degradation by proteases, and eventually cleave the fusion product to recover the desired protein or polypeptide. The selection of specific reagents for such cleavage has so far been very limited. One attractive way of cleavage has been the use of hydroxyla ine, which cleaves between the amino acids asparagine (Asn) and glycine (Gly) .

A drawback with hydroxylamine is, however, that in addition to cleaving the Asn-Gly bond, it also reacts with other asparagine and glutamine residues in the peptide or protein, resulting in the formation of hydroxamates (Canova-Davis, E. , et al., (1992) J. Biochem. 285, 207-

213) . These hydroxamates are difficult to separate from the "native" peptide or protein, and it is also difficult to analyse the content of hydroxamates in a peptide or protein product, since the formation of a hydroxamate of the peptide or protein only represents a mass change of 16 daltons an no change in charge.

The object of the present invention is to provide a cleaving agent which is devoid of the above disadvantages. In accordance with the present inventive concept, such a cleaving agent would, on one hand, like hydroxylamine, act as a nucleophile in the desired cleavage reaction, but , would, on the other hand, (i) be heavier than hydroxylamine such that, in the case of side reactions similar to the hydroxamate formation, these side reaction products would be easier to detect, and (ii) carry a charge such that the reaction products would be easier to separate.

According to the present invention, it has been found that specific cleavage of Asn-Gly bonds in proteins and

peptides may be effected by a method involving the use of a compound of the following general formula I:

R-l - (CH ₂ )n - NH - (CH ₂ ) _m - R2 (')

wherein

R _x is NH ₂ or OH,

R ₂ is hydrogen, lower alkyl, NH ₂ , OH or halo, n is an integer from 1 to 3, and m is 0 or an integer from 1 to 3, preferably from 1 to 2.

Lower alkyl has from 1 to 4 carbon atoms, such as methyl, ethyl, propyl and butyl.

Halo is fluoro, chloro, bromo or iodo, R-L is preferably NH ₂ .

R ₂ is preferably hydrogen, methyl, NH ₂ , OH, fluoro, chloro or bromo.

Preferred compounds of Formula I are ethylenediamine, 2-(ethylamino)ethylamine and 2-(2-aminoethylamino)ethanol. These compounds are commercially available or may be prepared by conventional methods, for example as described in Beilstein 4.286 and 4.IV.1174. The other compounds of Formula I are either commercially available or may be prepared by conventional methods, for example analogously with those mentioned above.

The compounds of Formula I are fairly strong nucleophiles, and if they cause reamidation on asparagine or glutamine, this will cause a net charge of +1 and a considerable mass change, e.g. of +43 for ethylenediamine. The cleavage reaction is preferably performed in a polar solvent, especially water or aqueous ethanol.

The compound of Formula I is usually dissolved in the reaction medium, but may also be immobilized to a solid phase. The protein or peptide to be cleaved according to the method of the invention contains an Asn-Gly bond at the desired cleavage site(s) . In the case of a recombinantly produced fusion protein, such as produced by recombinant

DNA techniques, a unique Asn-Gly bond is preferably present in the joint between the desired protein or peptide and the fusion partner. An example of such a fusion protein is that described by Moks, T., et al., (1987) Bio/Technology 5 , 379-382 as Z-IGF-I and consisting of a modified IgG-binding domain of Protein A fused to human insulin-like Growth Factor I (IGF-I) . In this fusion product, the N-terminal glycine of IGF-I is preceded by an asparagine residue.

The method of the invention is illustrated further in the following non-limiting examples.

EXAMPLE 1 Cleavage of apolipoprotein Apolipoprotein, Milanoform (a di er in which Arg in position 173 is changed to Cys), Mw 6530.4, hereinafter for brevity Apo, was obtained from Pharmacia AB, Sweden. The protein has an Asn-Gly sequence in positions 184-185, which was cleaved with (A) hydroxylamine and (B) ethylenediamine as described below.

A. Hydroxylamine cleavage (comparative) 100 μg of Apo were treated in 200 μl of 2 M hydroxylamine (Fluka, Schweiz) , 0.2 M Trisbase (Merck, Germany), 1.5 M NaOH, 1 mM EDTA, 10 % ethanol, 3 M guanidine-HCl, pH 9.5, at 45 °C for 6 to 24 h.

The cleavage mixture was subjected to analytical high performance liquid chromatography (HPLC) on a Beckman

System Gold HPLC (Beckman, USA) and a Brownlee Aquapore Butyl column, 7 μm, 220 x 4,6 mm (Applied Biosystems, USA) with a gradient 10-60 % B in A over 40 min. at 40 °C, where A is: 0.25 % pentafluoropropionic acid (PFPA) (Lancaster Synthesis, England) in MilliQ water, and B is: 0.25% PFPA in acetonitrile (HPLC guality; Baker, Holland) . The flow was 1.0 ml/min..

Two peaks were obtained, the first eluting peptide being identified as fragment 185-243 of Apo. B. Ethylenediamine cleavage (method of invention) 100 μg of Apo were treated in 125 μl of 1 M ethylenediamine (Fluka, Schweiz), 0.2 M Tris-base (Merck,

Germany) , 1 mM EDTA, 10 % ethanol, 3 M guanidine-HCl, pH 9.4, at 45 °C for 6 to 18 h.

Analytical HPLC was then performed in the same way as for the hydroxylamine cleavage mixture above and resulted in the same fast eluting peak (Apo fragment 185-243) being obtained.

EXAMPLE 2 Cleavage of Insulin-like Growth Factor I Insulin-like Growth Factor 1, hereinafter IGF-I, was produced in E. coli as a fusion protein Z-IGF-I, where Z is a modified domain of Staphylococcus protein A, essentially as described in Moks, T., et al., (1987) Bio/Technology 5_, 379-382. The cleavage site between Z and IGF-I is Asn-Gly, which site was cleaved with (A) hydroxylamine and (B) ethylenediamine as described below.

A. hydroxylamine cleavage (comparative)

Cleavage with hydroxylamine of 100 μg of Z-IGF-I was performed under the conditions specified for the corresponding cleavage of Apo in Example 1 above. After cleavage, IGF-I was purified by desalting to 0.2 M acetic acid on PD10 columns (Pharmacia LKB Biotechnology AB, Sweden) .

Desalting was followed by cation exchange on a MonoS column no. 2623 (Pharmacia LKB Biotechnology AB, Sweden) . The gradient used was 10-52 % B in A for 20 minutes, where A is: 20 mM ammonium acetate, 10% ethanol, pH 5.4, and B is: 1 M ammonium acetate, 10 % ethanol, pH 5.4.

Analytical HPLC was then performed on a Hewlett Packard 1090 using a YMC-Pack Protein-RP column, PRRP-03-5, C4, 5 μm, 250 x 4.6 mm (YMC Inc., USA) and a gradient of

30-40 % B in A over 40 min. at ambient temperature, where A is: 0.25 % PFPA in MilliQ water, and B is: 0.25 % PFPA in acetonitrile (HPLC quality). The flow was 1.0 l/min..

B. Ethylenediamine cleavage (method of invention) 100 μg of Z-IGF-I were cleaved with ethylenediamine under the same conditions as those specified for the ethylenediamine cleavage of Apo in Example 1.

Samples were taken after 2, 4 and 6 h and directly analysed, after desalting and cation exchange as described above for the hydroxylamine cleavage of Z-IGF-I, by HPLC on a Beckman System Gold HPLC (Beckman, USA) and a Brownlee Aquapore Butyl column, 7 μm, 220 x 4,6 mm (Applied

Biosystems, USA) with a gradient 26-31 % B in A over 30 min. at 50 °C, where A is: 0.25 % PFPA in MilliQ water, and B is: 0.25 % PFPA in acetonitrile (HPLC quality). The flow was 1.0 ml/min.. IGF-I was detected after 2 h, and at 4 h the IGF-I peak had increased by about two times. The IGF-I peak at 6 h was about the same size as that for the sample taken at 4 h.

The yield of ethylenediamine cleavage after 4 h was 25 % compared with that obtained in the optimized hydroxylamine cleavage.

Pure IGF-I was added to the 4 h sample, which was then analysed by HPLC. The IGF-I peak increased as assumed.

IGF-I from the ethylenediamine cleavage was isolated by semipreparative HPLC in the same way as for the analytical HPLC described above. The IGF-I fraction produced was then N-terminal sequenced in five steps by Edman degradation on a solid phase sequencer, Prosequenator 6600 (Millipore, USA) . Carboxyl groups on the peptides were covalently bound to arylamine membrane (Millipore) prior to sequencing. The sequence obtained was identical with that of IGF-I, NH2-GlyProGluThrLeu and no additional sequences could be detected.

The molecular weight of the peptides were determined on a plasma desorption time of flight mass spectrometer, PDMS (Perkin Elmer, USA, Biolon, Sweden) or on an ESR spectrometer (VG Quattro, Fisons, England) . The resulting mass spectra gave a single peak at Mw 7646 (theor. Mw 7649) . No peak at Mw 7690 was detected which should have been the case if ethylenediamine had reamidated with asparagine or glutamine.

EXAMPLE 3 Cleavage of Z-IGF-I with 2-ethylaminoethylamine

100 μg of the fusion protein Z-IGF-I were cleaved with 2-ethylaminoethylamine (Mw 88.15) under the same conditions as for the ethylenediamine cleavage of Z-IGF-I described in Example 2, except that the reaction temperature was decreased to 34 °C, CAPSO buffer, pH 9.3, (Sigma, USA) was used instead of Tris, and the reaction time was prolonged to 18 h. Analysis by HPLC on a Hewlett Packard 1090, using a

YMC-Pack Protein-RP column, PRRP-03-5, C4, 5 μm, 250 x 4.6 mm (YMC Inc. , USA) and a gradient 30-40 % B in A, where A is: 0.25 % PFPA in MilliQ water, and B is: 0.25 % PFPA in acetonitrile (HPLC quality) , over 30 min. at ambient temperature and a flow of 1.0 ml/min. indicated a yield of 45 % compared to hydroxylamine cleavage.

At these conditions, the yield of IGF-I, cleaved with ethylenediamine, was 32 % compared to hydroxylamine.

IGF-I from the cleavage reaction was purified via gel filtration, anion exchange and reversed phase HPLC. The mass spectra was identical to that of IGF-I and no peak at 7690 corresponding to reamidation could be detected.

EXAMPLE 4 Cleavage of Z-IGF-I with 2-(2--_minoethyla_nino)ethanol Cleavage of Z-IGF-I with 2-(2-aminoethylamino)ethanol at the same conditions as in Example 3 gave a yield of 28 % compared to hydroxylamine cleavage. Mass spectra after purification of IGF-I as in Example 3 was identical to that of IGF-I and no reamidation products could be detected.