Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-12-21
2001-02-27
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S024000, C435S069100, C435S183000, C435S219000, C435S212000, C435S320100, C435S252300, C435S068100, C435S019000, C536S023200, C536S023740, C536S023700, C530S350000
Reexamination Certificate
active
06194190
ABSTRACT:
This application is the national phase under 35 U.S.C. §371 of prior PCT International Application No. PCT/JP97/02121 which has an International filing date of Jun. 19, 1997 which designated the United States of America.
TECHNICAL FIELD
The present invention pertains to an amino terminal protecting group-releasing enzyme which has an activity for releasing a protecting group which is present at an amino terminal of a protein or a peptide. The present invention also pertains to a DNA encoding the above amino terminal protecting group-releasing enzyme. The present invention also pertains to a method for producing the above amino terminal protecting group-releasing enzyme by means of a genetic recombinant technique. The present invention also pertains to a method for removing an amino terminal protecting group by using the above amino terminal protecting group-releasing enzyme. The present invention further pertains to a method for analyzing an amino acid sequence by means of the above removal method. The present invention further pertains to a kit for use in analyzing an amino acid sequence, the kit comprising the above amino terminal protecting group-releasing enzyme. The present invention further pertains to an antibody or a fragment thereof specifically binding to the above amino terminal protecting group-releasing enzyme, or a functional equivalent thereof. The present invention further pertains to a synthesized oligonucleotide probe or a synthesized oligonucleotide primer which is capable of hybridizing to the above DNA, respectively.
BACKGROUND ART
The determination of the amino terminal amino acid sequences of proteins and peptides is an essential analysis for their identification or confirmation.
However, 60% or more of the soluble proteins derived from eukaryote reportedly have the &agr;-amino group at their amino terminal blocked by either an acetyl group or another protecting group; and in the Edman degradation method, a well-established commonly used method for. analyzing an amino acid sequence from the amino terminal, proteins and peptides of which amino terminal is blocked by a protecting group cannot be analyzed.
As the protecting groups blocking the amino terminal of proteins and peptides, formyl group, acetyl group, myristoyl group, pyroglutamyl group, dimethyl group, glucuronyl group, glycosyl group and trimethyl group have been reported. As the method for analyzing an amino terminal amino acid sequence for such blocked proteins, there can be employed the Edman degradation method applied after removing a protecting group.
The removal of protecting groups includes a method using an enzyme and a chemical method. However, an enzymatic method has a drawback of a lack of versatility, since different enzymes are used depending on the kinds of protecting groups to be removed. For example, acylamino acid releasing enzyme is used in the case of acetyl groups, and pyroglutamyl peptidase in the case of pyroglutamyl groups. Also, as to chemical method, no versatile methods have been developed to date. In addition, because there is no known method of identifying the protecting group when the amino terminal is blocked thereby, when the protecting group is actually removed, there are no other alternatives except that a number of methods for removing protecting group depending on respective protecting groups are attempted one by one. Furthermore, since there is no effective method for removing a protecting group available for the proteins and peptides blocked with myristoyl group, it is impossible to determine their amino acid sequences from the amino terminal.
Incidentally, as the peptidases isolated from
Pyrococcus furiosus
, the peptidases acting on an amino terminal portion of a peptide, there have been known an aminopeptidase (Japanese Patent Laid-Open No. Hei 6-319566), a pyroglutamyl peptidase (Japanese Patent Laid-Open No. Hei 7-298881), and a methionine aminopeptidase (Japanese Patent Laid-Open No. Hei 8-9979). Among these peptidases, the pyroglutamyl peptidase possesses an activity for releasing of the amino terminal pyroglutamyl group, but does not act on other protecting groups, e.g., acetyl groups. In addition, the other two kinds of enzymes both cannot act on amino terminal blocked by protecting groups.
As described above, the existing method for analyzing amino terminal amino acid sequence has the drawback of a lack of versatility when applied to proteins and peptides of which amino terminal is blocked by protecting groups.
DISCLOSURE OF INVENTION
Accordingly, an object of the present invention is to provide an amino terminal protecting group-releasing enzyme, the enzyme exhibiting an amino terminal group-releasing activity for two or more protecting groups, or a functional equivalent thereof; a DNA encoding the above enzyme; a method for producing the above enzyme; a method for removing an amino terminal protecting group comprising acting with the above enzyme; a method for analyzing an amino acid sequence by using the above method; and a kit for use in analyzing an amino acid sequence, the kit comprising the above enzyme. A further object of the present invention is to provide an antibody or a fragment thereof specifically binding to the above amino terminal protecting group-releasing enzyme, or a functional equivalent thereof; and a synthesized oligonucleotide probe or a synthesized oligonucleotide primer which is capable of hybridizing to the above DNA, respectively.
The present inventors have screened cosmid protein libraries from
Pyrococcus furiosus
, and have obtained a cosmid clone expressing an activity for releasing a protecting group at an amino terminal. The present inventors have isolated a gene of an amino terminal protecting group-releasing enzyme contained in this clone, and have determined its base sequence. In addition, the present inventors have constructed a recombinant plasmid for mass-expressing the enzyme in microorganisms. As a result, the present inventors have succeeded in producing the enzyme and have clarified various enzymological properties of the enzyme. Furthermore, the present inventors have found that the enzyme possesses an activity for releasing a plurality of amino terminal protecting groups such as typically an acetyl group. In addition, the present inventors have succeeded in preparing the functional equivalent of the enzyme. The present invention has been thus completed.
Specifically, in sum, the present invention pertains to:
[1] an amino terminal protecting group-releasing enzyme characterized in that the enzyme possesses an activity for releasing a protecting group by acting on a peptide of which amino terminal is blocked by the protecting group (hereinafter abbreviated as a term “amino terminal protecting group-releasing activity”), and exhibits the activity for two or more protecting groups;
[2] the enzyme according to the above item [1], wherein the enzyme exhibits an amino terminal protecting group-releasing activity for at least two or more of protecting groups selected from the group consisting of acetyl group, pyroglutamyl group, formyl group and myristoyl group;
[3] the enzyme according to the above claim
1
or
2
, wherein the enzyme further possesses an amino peptidase activity;
[4] the enzyme according to any one of the above items [1] to [3], wherein the enzyme possesses the following physicochemical properties:
(1) optimal temperature: 75° to 95° C. at a pH of 7.6;
(2) optimal pH: a pH of 6.5 to 9.5; and
(3) effects of various reagents:
the activity being inhibited by amastatin, and enhanced by CoCl
2
.
[5] the enzyme according to any one of the above items [1] to [4], wherein the enzyme comprises an entire sequence of the amino acid sequence as shown by SEQ ID NO: 1 in Sequence Listing, or a partial sequence thereof, and wherein the enzyme exhibits an amino terminal protecting group-releasing activity;
[6] a functional equivalent of the enzyme according to the above item [5], wherein
Izu Yukiko
Kato Ikunoshin
Miyagi Masaru
Tanaka Tetsuki
Tanigawa Tetsuo
Birch & Stewart Kolasch & Birch, LLP
Hutson Richard
Prouty Rebecca E.
Takara Shuzo Co. Ltd.
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