Thermostable peptidase

Details Thermostable peptidase Thermostable peptidase

1999-06-15

2001-09-25

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S320100, C435S325000, C435S252300, C435S006120, C435S455000, C435S044000, C536S023200

Reexamination Certificate

active

06294367

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been identified as a thermostable peptidase.
BACKGROUND OF THE INVENTION
Hyperthermophiles are microorganisms which grow optimally at temperatures of 80° C. and above (Huber et al.,
J. Biotechnol.
64, 39-52 (1998)). A majority of these organisms fall under the newly identified domain, archaea (Woese et al.,
Proc. Nat. Acad. Sci USA
87, 4576-4579 (1990)). One of the most extensively studied hyperthermophilic archaea is
Pyrococcus furiosus
(Pfu). This microorganism has a growth optimum of 100° C. (Fiala et al.,
Arch. Microbiol.
145, 56-61 (1986)). It is a heterotrophic, anaerobic archaeon, and utilizes complex carbohydrates and peptides/proteins as carbon and energy sources (Adams et al.,
Adv. Protein Chem.
48, 101-180 (1996)). The metabolic end products are organic acids, alanine, CO
2
and H
2
(Kengen et al.,
Arch. Microbiol.
161, 168-175 (1994); Kengen et al.,
FEMS Microbiol.
Rev. 18, 119-137 (1996)). Additional energy is generated when excess redox equivalents are channeled to elemental sulfur (Schicho et al.,
J. Bacteriol.
175, 1823-1830 (1993)).
Most of the proteins isolated from these hyperthermophiles exhibit a temperature optimum of at least 80-100° C. or above (Adams et al.,
Bio/Technology
13, 662-668 (1995); Adams et al.,
Trends Biotechnol
16, 329-332 (1998)). Accordingly, there is much interest in exploiting these proteins for biotechnological applications, as they are able to perform biochemical reactions under harsh conditions, such as in the presence of high-temperatures, organic solvents, and denaturants (Adams et al., supra.)
P. furiosus
has been the source of many of these biotechnologically important proteins, including DNA polymerase (Lundberg et al.,
Gene
108, 1-6 (1991)), &agr;-amylase (Laderman et al.,
J. Biol. Chem.
268, 24394-24401 (1993)), and proteases (Voorhorst et al.,
J. Biol. Chem.
271, 20426-20431 (1996); Harwood et al.,
J. Bacteriol.
179, 3613-3618 (1997)).
Peptidases hydrolyze peptide bonds from peptide and protein molecules, For example, carboxypeptidases sequentially hydrolyze peptide bonds from the C-terminus of proteins and polypeptides. They are ubiquitous in animals, plants and microorganisms; many carboxypeptidases have been characterized based on their substrate specificity and mechanism (serine- versus metallo-carboxypeptidase) (Skidgel et al.,
Immunol. Rev.
161, 129-141 (1998)). Carboxypeptidases have been implicated in physiological roles such as protein degradation/turnover, or processing of precursor proteins (Steiner, D. F.,
Curr. Opin. Chem. Biol.
2, 31-39 (1998)), and in the metabolism of proteins and peptides as carbon or energy sources. Most of the purified carboxypeptidases have a temperature optimum below 40° C. However, three moderately thermostable carboxypeptidases have been purified from the bacteria
Thermoactinomyces vulgaris
(Stepanov et al., T. Methods Enzymol. 248, 675-683 (1995)), and
Thermus aquaticus
(Lee et al., Biosci. Biotechnol. Biochem. 56, 1839-1844 (1992)), and the archaeon
Sulfolobus solfataricus
(Colombo et al., Eur. J. Biochem. 206, 349-357 (1992)), with temperature optima of 60, 80, and 85° C., respectively. Carboxypeptidase activity has not yet been reported for
P. furiosus.
Protein sequencing is an integral component of modern biochemical research. Edman degradation is useful for N-terminal sequencing, but it fails when the amino terminus is chemically protected. Aside from endoproteolytic fragmentation, another way to obtain sequence information from proteins is to sequence from the C-terminus. Various C-terminal sequencing methods have been developed: chemical cleavage analogous to Edman degradation (Hardeman et al., Protein Sci. 7, 1593-1602 (1998)), and enzymatic digestion by carboxypeptidases (Thiede et al., Eur. J. Biochem. 244, 750-754 (1997)). A particularly powerful approach is enzymatic ladder sequencing, in which a carboxypeptidase is used to generate a set of differentially cleaved peptides that can be visualized in a mass spectrum; mass differences between adjacent peaks correspond to the molecular masses of individual amino acids that have been released. Enzymatic protein ladder sequencing has the potential to sequence as far as the enzyme can cut. However, a number of difficulties have limited the applicability of this approach: i) the limited specificities of a given carboxypeptidase toward the 20 common amino acids; and ii) the resistance of native protein molecules to digestion at mesophilic temperatures. There is a need for novel peptidase enzymes having enhanced thermostability. This includes a need for thermostable C-terminal peptidases whose enhanced thermostability is beneficial in sequencing reactions.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compositions and methods useful in C-terminal sequencing of proteins and polypeptides. A hyperthermophilic carboxypeptidase of the present invention is catalytically active at temperatures where the tertiary structures of most mesophilic proteins are denatured. Accordingly, the use of the carboxypeptidase of the invention provides distinct advantages over previous peptidases used in sequencing of polypeptides. Additionally, the specificity of a thermophilic carboxypeptidase of the invention is broader than most of its mesophilic counterparts.
In one embodiment the present invention provides a substantially purified polypeptide characterized as a thermostable carboxypeptidase. The carboxypeptidase is a metallocarboxypeptidase and has a monomeric molecular weight of about 58 kDa by SDS-PAGE; a monomeric molecular weight of about 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; and a dimeric molecular weight of about 128 kDa by gel filtration chromatography. In another aspect of the invention the carboxypeptidase activity is dependent on the presence of the divalent cation (e.g., Co
2+
, Ca
2+
, Ni
2+
, Mn
2+
, Fe
2+
, Cd
2+
, Cu
2+
, Pb
2+
, Rh
2+
). The purified carboxypeptidase can be obtained from the genus Pyrococcus. A preferred source is
P. furiosus.
In yet a further embodiment, the polypeptide of the invention has a sequence as set forth in SEQ ID Nos 1, 2, or 3.
In another embodiment the present invention provides a polynucleotide encoding a thermostable carboxypeptidase, as well as vectors containing the polynucleotide and host cells containing a vector with a polynucleotide encoding a thermostable carboxypeptidase.
In yet another embodiment, the present invention provides an anti-polypeptide antibody that binds to a thermostable carboxypeptidase of the invention. The antibody may be monoclonal or polyclonal.
In another embodiment, a process for producing a polypeptide characterized as a thermostable carboxypeptidase is provided. The process includes expressing from a host cell a thermostable carboxypeptidase polypeptide encoded by a polynucleotide of the invention.
Also provided is a process for producing a cell by transforming or transfecting the cell with a vector containing a polynucleotide encoding a thermostable carboxypeptidase such that the cell expresses the polypeptide encoded by the polynucleotide.
The present invention also provides a method for sequencing a polypeptide's amino acid sequence, comprising contacting a polypeptide with a thermostable carboxypeptidase having a temperature optimum exceeding 90° C., wherein the carboxypeptidase hydrolyzes peptide bonds in the polypeptide; and identifying the enzymatic products.
These and other aspects of the present invention will be apparent to those of skill in the art from the teachings herein.


REFERENCES:
Davison et al., Virology, 186, 9-14, Feb. 1992.*
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