Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-09
2001-10-30
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S069520, C530S300000, C530S311000, C530S331000, C530S343000, C530S350000, C530S351000, C530S399000
Reexamination Certificate
active
06309859
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for removing an optionally oxidized methionine residue present at the N-terminal of a peptide (including a protein) or a salt thereof.
PRIOR ART
When a protein is synthesized within a cell, its amino terminus is always occupied by methionine, which corresponds to the mRNA initiation condon, i.e. AUG. In naturally produced mature protein molecules, however, that methionine residue is usually no longer present, because it is removed via subsequent processing.
Recent progress in gene recombination technology has made it possible to produce useful proteins using microbes or animal cells, e.g.
Escherichia coli
. In some cases the protein produced retains the methionine described above. For example, in human growth hormone expressed by
Escherichia coli
, the methionine addition rate reaches approximately 100% [Nature, 293, 408 (1981)], and 50% in interferon-&agr; [Journal of Interferon Research, 1, 381 (1981)]. In the production of non-glycosylated human interleukin-2 (rhIL-2) expressed by
Escherichia coli
, in addition to molecule whose initiation amino acid residue is alanine, like natural human interleukin-2, a second molecule with a methionine residue added at the amino terminus (Met-rhIL-2) was found.
Dixon reported in 1964 that DL-Ananylglycine when reacted with glyoxylic acid, pyridine and cupric acetate produced pyruvoylglycine by transamination [Biochemistry Journal, 92, 661 (1964)]. Moreover, Dixon reported that thus obtained pyruvoylglycine was reacted with thiosemicarbazide to produce glycine by splitting an amide linkage [Biochemistry Journal, 90, 2C (1964)]. Furthermore, Dixon reported that the above described chemical reaction was applied to Pseudomonas cytochrome C-551 and the N-terminal glutamic acid was removed [Biochemistry Journal, 94, 463 (1965)].
However, the reactions reported by Dixon relate to removal of a N-terminal amino acid of synthesized peptide or mature protein and there is no report in the more than 30 years following this report that the above described chemical reaction might be useful for removal of the N-terminal methionine added to a protein produced by gene recombination technology.
It can be hypothesized that the three dimensional structure, bioactivity and stability may differ between a molecule with methionine at its amino terminus and one without methionine, even though both molecules are otherwise the same protein; addition of methionine at the amino terminus is believed to possibly cause an increase in protein antigenicity. Therefore, it would be important, in industrial application, to establish a relatively simple and efficient method of selectively removing such amino terminal methionine.
In prior methods for solving this problem, a process was suggested by which methionine could be removed by cyanogen bromide (BrCN) cleavage [Science, 198, 1056 (1977)]; however, no satisfactory result has been obtained, since the process not only premises the absence of other methionine residue(s) in the molecule of the required mature protein but also subjects the protein to a drastic chemical reaction.
A chemical method which makes it possible to remove a N-terminal methionine residue selectively and efficiently from a peptide or protein having a methionine residue at its N-terminal irrespective of kinds of peptide or protein is presently unknown. This fact is thought to be caused by the difficulty in finding an appropriate chemical reaction for removing a N-terminal methionine under mild conditions without denaturing a peptide or protein which is a final product. In particular, in case of the removing an extra N-terminal methionine added to a protein which has a relatively high molecular weight, and is produced by gene recombination technology particularly, aimed for a medical use, it is required not to lower an activity of the protein after removal of the methionine. Thus, it is usually necessary to proceed such reaction in a weakly acidic to basic solution without heating. Therefore, in the state of the arts, good reaction conditions have not been reported, since a lot of limitations are required for the chemical reaction.
SUMMARY OF THE INVENTION
The present inventors, after studying how to provide a method for producing a protein having a natural type amino acid sequence by selectively removing a N-terminal methionine in a protein produced by gene recombination technology, found a method for removing a N-terminal methionine from a protein having an additional methionine by converting a methionine in the protein having the additional methionine to an &agr;-diketone and further by reacting the &agr;-diketone with an organic diamine derivative, thereby, obtaining a peptide or protein having no additional methionine at its N-terminal. The present method for obtaining the peptide or protein is characterized by reacting a peptide or protein having an additional methionine represented by the following formula (I) with a glyoxylic acid as an &agr;-diketone derivative, a cupric sulphate being capable of giving a transition metal ion and a pyridine as an amine derivative to carry out transamination and convert methionine to &agr;-diketone, followed by subjecting the obtained &agr;-diketone derivative to hydrolysis with an o-phenylenediamine as a diamine derivative and also characterized by removing the N-terminal methionine from the peptide or protein having the additional methionine without lowering the activity of the peptide or protein.
The finding was followed by further research, leading to the completion of the present invention.
In the formula (I), X represents either an amino acid residue or a peptide chain having two or more amino acids. Preferably, X represents a peptide chain which is a part of a protein produced by gene recombination technology. Furthermore, the peptide or protein consisting of multiple amino acids may be either glycosylated or non-glycosylated one.
That is, the present invention relates to
(1) a method for removing a N-terminal optionally oxidized methionine residue, which comprises reacting a peptide or a salt thereof having said methionine residue at its N-terminal with an &agr;-diketone derivative and then subjecting the obtained product to hydrolysis;
(2) a method of the above (1), wherein the peptide having an optionally oxidized methionine residue at its N-terminal is a protein produced by gene recombination technology;
(3) a method of the above (2), wherein the peptide produced by gene recombination technology has at least 30 amino acid residues. One preferred group of peptides is growth hormone, neurotrophin-3, betacellulin, parathyroid hormone or interleukin-2 each having an optionally oxidized methionine residue at its N-terminal;
(4) a method of the above (1), wherein the &agr;-diketone derivative is reacted in the presence of a transition metal ion;
(5) a method of the above (1), wherein the &agr;-diketone derivative is reacted in the presence of a base;
(6) a method of the above (1), wherein the &agr;-diketone derivative is reacted in the presence of a transition metal ion and a base;
(7) a method of the above (1), wherein the &agr;-diketone derivative is glyoxylic acid or a salt thereof;
(8) a method of the above (4), wherein the transition metal ion is a copper ion;
(9) a method of the above (5), wherein the base is a pyridine;
(10) a method of the above (1), wherein the hydrolysis is carried out with using a base;
(11) a method of the above (10), wherein the base is an amine derivative;
(12) a method of the above (10), wherein the base is a diamine derivative, or a thio- or seleno-semicarbazide derivative;
(13) a method of the above (12), wherein the diamine derivative is an o-phenylenediamine;
(14) a method for producing human growth hormone or a salt thereof, which comprises reacting human growth hormone or a salt thereof having a methionine at its N-terminal which is produced by gene recombination technology with a glyoxylic acid or a salt thereof in the presence of a
Nishimura Osamu
Ohmae Hiroaki
Suenaga Masato
Tsuji Shinji
Celsa Bennett
Conlin David G.
Dike Bronstein Roberts & Cushman IP Group
Takeda Chemical Industries Ltd.
LandOfFree
Method for removing N-terminal methionine does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for removing N-terminal methionine, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for removing N-terminal methionine will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2570727