# US 12,110,516 B2 · Thermostable terminal deoxynucleotidyl transferase

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- Number: US 12,110,516 B2
- Document ID: US12110516B2
- Status: Granted
- Kind: B2
- Application number: 17/317,721
- Filed: 2021-05-11
- Published: 2021-11-18
- Granted: 2024-10-08
- Earliest claimed: 2020-05-12
- Assignees: Illumina, Inc.; Illumina Singapore Pte. Ltd.; National University of Singapore
- Applicants: Illumina, Inc.; Illumina Singapore Pte. Ltd.; National University of Singapore
- Inventors: Saurabh Rajendra Nirantar; Jasmine Puay Suan Chua; Sergio Peisajovich; Wen Shan Yew; Maybelle Darlene Kho Go
- Web page: https://patentgrep.com/patent/US12110516B2

## CPC

- C12N 9/1264: {DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase}
- C12Y 207/07031: DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Disclosed herein include recombinant terminal deoxynucleotidyl transferases (TdTs). In some embodiments, the recombinant TdT comprises an amino acid sequence that is at least 80% identical to a bovine TdT, wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 in the bovine TdT.

## Claims

### Claim 1

A recombinant terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions corresponding to Glu191, Glu194, Asp242, Lys287, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12.

### Claim 2 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to Glu191 in the Bos taurus TdT of SEQ ID NO: 12 is Glu191Val.

### Claim 3 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to Glu194 in the Bos taurus TdT of SEQ ID NO: 12 is Glu194Gly.

### Claim 4 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to Asp242 in the Bos taurus TdT of SEQ ID NO: 12 is Asp242Tyr.

### Claim 5 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to Lys287 in the Bos taurus TdT of SEQ ID NO: 12 is Lys287Glu.

### Claim 6 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to Met299 in the Bos taurus TdT of SEQ ID NO: 12 is Met299Lys.

### Claim 7 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to Thr342 in the Bos taurus TdT of SEQ ID NO: 12 is Thr342Ser.

### Claim 8 (depends on 1)

The recombinant TdT of claim 1, wherein the amino acid substitution mutation at the position corresponding to His421 in the Bos taurus TdT of SEQ ID NO: 12 is His421Pro.

### Claim 9 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises two or more amino acid substitution mutations at two or more positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12.

### Claim 10 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises nine amino acid substitution mutations at positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12.

### Claim 11 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11.

### Claim 12 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12.

### Claim 13 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT is stable at a temperature of 47° C.or higher.

### Claim 14 (depends on 1)

The recombinant TdT of claim 1, wherein the terminal deoxynucleotidyl transferase activity of the recombinant TdT is at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120%, of the terminal deoxynucleotidyl transferase activity of the Bos taurus TdT of SEQ ID NO: 12 at a same test temperature.

### Claim 15 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises a small ubiquitin-like modifier (SUMO) fragment.

### Claim 16 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises the SUMO fragment on the N-terminus of the recombinant TdT.

### Claim 17 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

### Claim 18 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 1.

### Claim 19 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises three or more amino acid substitution mutations at three or more positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12.

### Claim 20 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises an amino acid substitution mutation at a position corresponding to Lys193 in the Bos taurus TdT of SEQ ID NO: 12.

### Claim 21 (depends on 20)

The recombinant TdT of claim 20, wherein the amino acid substitution mutation at the position corresponding to Lys193 in the Bos taurus TdT of SEQ ID NO: 12 is Lys193Asn.

### Claim 22 (depends on 1)

The recombinant TdT of claim 1, wherein the recombinant TdT comprises an amino acid substitution mutation at a position corresponding to Phe296 in the Bos taurus TdT of SEQ ID NO: 12.

### Claim 23 (depends on 22)

The recombinant TdT of claim 22, wherein the amino acid substitution mutation at the position corresponding to Phe296 in the Bos taurus TdT of SEQ ID NO: 12 is Phe296Leu.