# US 10,766,923 B2 · Methods and compositions to increase the rate of ligation reactions catalyzed by a sortase

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- Number: US 10,766,923 B2
- Document ID: US10766923B2
- Status: Granted
- Kind: B2
- Application number: 16/072,155
- Filed: 2017-01-26
- Published: 2019-03-14
- Granted: 2020-09-08
- Earliest claimed: 2016-01-26
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Robert T. Clubb; Brendan R. Amer
- Web page: https://patentgrep.com/patent/US10766923B2

## CPC

- C07K 1/13: Labelling of peptides
- C07K 7/06: having 5 to 11 amino acids
- C07K 14/35: from Mycobacteriaceae (F)
- C07K 14/47: from mammals
- C12N 9/52: derived from bacteria {or Archaea}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12Y 304/2207: Sortase A (3.4.22.70)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/21: containing a His-tag
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- C07K 2319/50: containing protease site
- C07K 2319/90: containing a motif for post-translational modification
- C12N 2310/3181: Peptide nucleic acid, PNA
- C12N 2310/3517: Marker; Tag
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

In various embodiments methods and compositions to increase the rate and/or activity of ligation reactions catalyzed by a sortase. In certain embodiments reagents are provided that comprises, inter alia, a polypeptide comprising an amino terminal polyglycine sequence comprising at least three contiguous Gly residues (e.g., a triglycine sequence, a tetraglycine sequence, a pentaglycine sequence, etc.) followed by a peptide that is to be ligated to a moiety followed by a sequence comprising the catalytic domain of a Sortase A enzyme.

## Claims

### Claim 1

A reagent for ligating a desired polypeptide to a target moiety having a linking peptide attached thereto where the linking peptide comprises an LPXTG (SEQ ID NO: 2) amino acid motif in the carboxyl terminal of the linking peptide, said reagent comprising:
a polypeptide comprising an amino terminal polyglycine sequence comprising at least three contiguous Gly residues sequence followed by said desired polypeptide sequence followed by a sequence comprising the catalytic domain of a Sortase A enzyme.

### Claim 2 (depends on 1)

The reagent of claim 1, wherein said polyglycine sequence comprises or consists of at least four contiguous Gly residues.

### Claim 3 (depends on 1)

The reagent of claim 1, wherein said polyglycine sequence comprises or consists of at least five contiguous Gly residues.

### Claim 4 (depends on 1)

The reagent of claim 1, wherein said reagent catalyzes a transpeptidation reaction substantially more rapidly and/or substantially more completely than a transpeptidation reaction performed using a sortase without an attached amino terminal pentaglycine.

### Claim 5 (depends on 1)

The reagent according to of claim 1, wherein a protease cleavage site is disposed between said desired polypeptide and said Sortase A enzyme.

### Claim 6 (depends on 5)

The reagent of claim 5, wherein said protease cleavage site comprises a protease site recognized by a protease selected from the group consisting of a serine protease, a cysteine or cysteine-like protease, and a metalloprotease.

### Claim 7 (depends on 5)

The reagent of claim 5, wherein said protease cleavage site comprises a protease site recognized by Ulp-1.

### Claim 8 (depends on 1)

The reagent of claim 1, wherein said reagent further comprises a carboxyl terminal affinity tag.

### Claim 9 (depends on 8)

The reagent of claim 8, wherein said affinity tag comprises a poly histidine tag.

### Claim 10 (depends on 8)

The reagent of claim 8, wherein said affinity tag comprises a His6 (SEQ ID NO:4) tag.

### Claim 11 (depends on 1)

The reagent of claim 1, wherein said catalytic domain of a sortase A enzyme comprise a catalytic domain of a Staphylococcus aureus sortase A enzyme.

### Claim 12 (depends on 1)

The reagent of claim 1, wherein said wherein said desired protein ranges in length from about 5 up to about 200 amino acids.

### Claim 13 (depends on 1)

The reagent of claim 1, wherein said desired protein comprises SUMO.

### Claim 14 (depends on 1)

The reagent of claim 1, wherein the amino acid sequence of said linking peptide comprises an amino acid sequence selected from the group consisting of LPETG (SEQ ID NO:18), LPATG (SEQ ID NO:19), LPKTG (SEQ ID NO:20), LPTTG (SEQ ID NO:21), LPSTG (SEQ ID NO:22), LPLTG (SEQ ID NO:23), and LPYTG (SEQ ID NO:24).

### Claim 15 (depends on 14)

The reagent of claim 14, wherein the amino acid sequence of said linking peptide consists of an amino acid sequence selected from the group consisting of LPETG (SEQ ID NO:18), LPATG (SEQ ID NO:19), LPKTG (SEQ ID NO:20), LPTTG (SEQ ID NO:21), LPSTG (SEQ ID NO:22), LPLTG (SEQ ID NO:23), and LPYTG (SEQ ID NO:24).

### Claim 16 (depends on 14)

The reagent of claim 14, wherein the amino acid sequence of said linking peptide comprises the amino acid sequence LPETG (SEQ ID NO:18).

### Claim 17 (depends on 16)

The reagent of claim 16, wherein the amino acid sequence of said linking peptide consists of the amino acid sequence LPETG (SEQ ID NO:18).