# US 6,294,358 B1 · Thermus promoters for gene expression

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- Number: US 6,294,358 B1
- Document ID: US6294358B1
- Status: Granted
- Kind: B1
- Application number: 09/390,867
- Filed: 1999-09-07
- Granted: 2001-09-25
- Earliest claimed: 1999-09-07
- Assignees: Thermogen, Inc.
- Inventors: Mikhail Peredultchuk; Veronica Vonstein; David C. Demirjian
- Web page: https://patentgrep.com/patent/US6294358B1

## CPC

- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a system for identifying, isolating and utilizing promoter elements useful for expression of nucleotide sequences and the proteins encoded thereby in a thermophile. In one embodiment, a recombinant DNA molecule is provided, and comprises a reporter sequence, a putative thermophile promoter, a selectable marker sequence, and a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile. Further, within the recombinant DNA, the reporter sequence is under the transcriptional control of a promoter which functions in a thermophile to form a promoter/reporter cassette, the promote/reporter cassette is flanked by said 3′ and said 5′ DNA targeting sequences, and the promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.

## Claims

### Claim 1

A recombinant expression vector comprising a thermophile promoter, said promoter comprising a DNA sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.

### Claim 2 (depends on 1)

The recombinant expression vector of claim 1 wherein the thermophile promoter sequence is operably linked to a reporter sequence.

### Claim 3 (depends on 1)

The recombinant expression vector of claim 1 wherein the thermophile promoter sequence is operably linked to a nucleic acid sequence encoding a protein.

### Claim 4

An isolated DNA molecule comprising the thermophile terminator sequence of SEQ ID NO: 13.

### Claim 5

A recombinant expression vector comprising:
a) a coding sequence encoding a protein;
b) a thermophile promoter selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44 operatively linked to said coding sequence to form a promoter/coding sequence cassette;
c) a drug resistance marker; and,
d) a 3′ and a 5′ DNA targeting sequence that are together capable of allowing integration of the promoter/coding sequence cassette into the genome of a thermophile;
wherein said promoter/coding sequence cassette is flanked by said 3′ and said 5′ DNA targeting sequences; and, said promoter/coding sequence cassette is positioned in the opposite orientation of the DNA targeting sequences.

### Claim 6 (depends on 5)

A method for expressing a protein in a bacterium of the genus Thermus comprising transforming into the bacterium the recombinant expression vector of claim 5 wherein the promoter/coding sequence cassette is stably integrated into the genome of the bacterium and culturing the transformed bacterium under culture conditions wherein the protein is expressed.

### Claim 7 (depends on 5)

A host cell having the promoter/coding sequence cassette of claim 5 stably integrated into its genome such that expression of the protein occurs within the host cell.

### Claim 8

A recombinant expression vector comprising a thermophile promoter wherein the thermophile promoter comprises SEQ ID NO: 11 and wherein the thermophile promoter sequence is operably linked to a reporter sequence.

### Claim 9

A recombinant expression vector comprising a thermophile promoter wherein the thermophile promoter comprises SEQ ID NO: 11 and wherein the thermophile promoter sequence is operably linked to a nucleic acid sequence encoding a protein.