# US 6,294,367 B1 · Thermostable peptidase

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- Number: US 6,294,367 B1
- Document ID: US6294367B1
- Status: Granted
- Kind: B1
- Application number: 09/333,768
- Filed: 1999-06-15
- Granted: 2001-09-25
- Earliest claimed: 1998-06-15
- Assignees: California Institute of Technology
- Inventors: Timothy C. Cheng; Vij Ramakrishnan; Sunney I. Chan
- Web page: https://patentgrep.com/patent/US6294367B1

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Thermostable peptidase enzyme derived from archaeon from the genus Pyrococcus is disclosed. The enzyme is produced from native or recombinant host cells and can be utilized in the biotechnology industry as a useful enzyme in sequencing reactions.

## Claims

### Claim 1

An isolated polynucleotide encoding a polypeptide, wherein said polypeptide comprises:
a) each sequence set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and
b) a thermostable metallocarboxypeptidase activity,
wherein said polynucleotide is derived from P. furiosus.

### Claim 2 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide is DNA.

### Claim 3 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide is RNA.

### Claim 4 (depends on 1)

A vector containing the polynucleotide of claim 1.

### Claim 5 (depends on 4)

The vector of claim 4, wherein the vector is an expression vector.

### Claim 6 (depends on 4)

The vector of claim 4, wherein the vector is a plasmid.

### Claim 7 (depends on 4)

The vector of claim 4, wherein the vector is a viral vector.

### Claim 8 (depends on 4)

A host cell containing the vector of claim 4.

### Claim 9 (depends on 8)

The host cell of claim 8, wherein the host cell is a prokaryotic cell.

### Claim 10 (depends on 8)

The host cell of claim 8, wherein the host cell is a eukaryotic cell.

### Claim 11 (depends on 8)

A process for producing a polypeptide comprising:
expressing from the host cell of claim 8 a polypeptide encoded by the polynucleotide.

### Claim 12 (depends on 4)

A process for producing a cell comprising: transforming or transfecting the cell with the vector of claim 4 such that the cell expresses the polypeptide encoded by the polynucleotide contained in the vector.

### Claim 13 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide comprises a monomeric molecular weight of about 58 kDa by SDS-PAGE.

### Claim 14 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide comprises a monomeric molecular weight of about 59 kDa as determined by matrix-assisted laser desorption ionization time- of-flight mass spectrometry.

### Claim 15 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide comprises a dimeric molecular weight of about 128 kDa by gel filtration chromatography.

### Claim 16 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide is a homodimer.

### Claim 17 (depends on 1)

The polynucleotide of claim 1, wherein the carboxypeptidase activity of the polypeptide is dependent upon the presence of a divalent cation.

### Claim 18 (depends on 17)

The polynucleotide of claim 17, wherein the divalent cation is selected from the group consisting of Co2+, Ca2+, Ni2+, Fe2+, Cd2+, Cu2+, Pb2+, Rh2+, Mn2+ and Zn2+.

### Claim 19 (depends on 1)

The polynucleotide of claim 1, wherein the thermostable carboxypeptidase activity of the polypeptide has a temperature optimum of at least 90° C.