# US 9,752,136 B2 · Polypeptides having endolysin activity and uses thereof

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- Number: US 9,752,136 B2
- Document ID: US9752136B2
- Status: Granted
- Kind: B2
- Application number: 14/818,927
- Filed: 2015-08-05
- Published: 2016-01-21
- Granted: 2017-09-05
- Earliest claimed: 2013-02-05
- Assignees: Plant Bioscience Limited
- Applicants: Plant Bioscience Limited
- Inventors: Melinda Mayer; Arjan Narbad
- Web page: https://patentgrep.com/patent/US9752136B2

## CPC

- C12N 9/2462: {Lysozyme (3.2.1.17)}
- A23K 50/00: Feeding-stuffs specially adapted for particular animals
- A23K 50/30: for swines
- A23K 50/40: for carnivorous animals, e.g. cats or dogs
- A23K 50/75: for poultry
- A23K 50/80: for aquatic animals, e.g. fish, crustaceans or molluscs
- C12N 9/80: acting on amide bonds in linear amides {(3.5.1)}
- C12Y 305/01028: N-Acetylmuramoyl-L-alanine amidase (3.5.1.28)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12Y 302/01017: Lysozyme (3.2.1.17)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23K: FODDER
- C12Y: ENZYMES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to novel polypeptides derived from endolysins from a bacteriophage of Clostridium perfringens and to nucleic acid molecules encoding the same, as well as compositions thereof. The invention also provides uses of such polypeptides and nucleic acid molecules in the diagnosis and treatment of conditions and diseases associated with microbial cells such as Clostridium perfringens. In particular, the invention provides a polypeptide having endolysin activity derived from bacteriophage ΦCP51 of Clostridium perfringens and uses thereof.

## Claims

### Claim 1

An isolated nucleic acid molecule encoding a fusion polypeptide,
wherein the fusion polypeptide comprises: i) a polypeptide having endolysin activity and comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 18 to 134 of SEQ ID NO: 1; and ii) a polypeptide that is heterologous to the polypeptide of i).

### Claim 2 (depends on 1)

The nucleic acid molecule according to claim 1 wherein the nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 2.

### Claim 3 (depends on 1)

A vector comprising the nucleic acid molecule according to claim 1.

### Claim 4 (depends on 3)

The vector according to claim 3 wherein the vector is an expression vector.

### Claim 5 (depends on 3)

The vector according to claim 3 wherein the vector is selected from the group consisting of pET15b and pACY184.

### Claim 6 (depends on 1)

A bacterial host cell comprising the nucleic acid molecule according to claim 1.

### Claim 7 (depends on 6)

The host cell according to claim 6 wherein the host cell is capable of expressing the fusion polypeptide.

### Claim 8 (depends on 6)

The host cell according to claim 6 wherein the host cell is non-pathogenic.

### Claim 9 (depends on 6)

The host cell according to claim 6 wherein the host cell is selected from the group consisting of cells of Escherichia coli, Lactococcus sp., Bacteroides sp., Lactobacillus sp., Enterococcus sp. and Bacillus sp.

### Claim 10 (depends on 8)

The host cell according to claim 8 wherein the host cell is a Lactococcus lactis or Lactobacillus johnsonii cell.

### Claim 11 (depends on 10)

The host cell according to claim 10 wherein the host cell is a Lactococcus lactis cell selected from the group consisting of L. lactis FI10676 and L. lactis UKLc10.

### Claim 12 (depends on 10)

The host cell according to claim 10 wherein the host cell is a Lactobacillus johnsonii cell selected from the group consisting of L. johnsonii F19785 and L. johnsonii FI10744.

### Claim 13

An expression vector comprising a heterologous nucleic acid molecule encoding a polypeptide, wherein the polypeptide has endolysin activity and comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 18 to 134 of SEQ ID NO: 1.

### Claim 14 (depends on 13)

The expression vector according to claim 13 wherein the expression vector is selected from the group consisting of pET15b and pACY184.

### Claim 15 (depends on 13)

A bacterial host cell comprising the expression vector according to claim 13.

### Claim 16 (depends on 15)

The host cell according to claim 15 wherein the host cell is capable of expressing the polypeptide.

### Claim 17 (depends on 15)

The host cell according to claim 15 wherein the host cell is non-pathogenic.

### Claim 18 (depends on 15)

The host cell according to claim 15 wherein the host cell is selected from the group consisting of cells of Escherichia coli, Lactococcus sp., Bacteroides sp., Lactobacillus sp., Enterococcus sp. and Bacillus sp.

### Claim 19 (depends on 18)

The host cell according to claim 18 wherein the host cell is a Lactococcus lactis or Lactobacillus johnsonii cell.

### Claim 20 (depends on 19)

The host cell according to claim 19 wherein the host cell is a Lactococcus lactis cell selected from the group consisting of L. lactis FI10676 and L. lactis UKLc10.

### Claim 21 (depends on 19)

The host cell according to claim 19 wherein the host cell is a Lactobacillus johnsonii cell selected from the group consisting of L. johnsonii FI9785 and L. johnsonii FI10744.

### Claim 22

A non-pathogenic bacterial host cell comprising a heterologous nucleic acid molecule encoding a polypeptide, wherein the polypeptide has endolysin activity and comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 18 to 134 of SEQ ID NO: 1.

### Claim 23 (depends on 22)

The host cell according to claim 22 wherein the nucleic acid molecule comprises an expression vector selected from the group consisting of pET15b and pACY184.

### Claim 24 (depends on 22)

The host cell according to claim 22 wherein the host cell is capable of expressing the polypeptide.

### Claim 25 (depends on 22)

The host cell according to claim 22 wherein the host cell is selected from the group consisting of cells of Escherichia coli, Lactococcus sp., Bacteroides sp., Lactobacillus sp., Enterococcus sp. and Bacillus sp.

### Claim 26 (depends on 25)

The host cell according to claim 25 wherein the host cell is a Lactococcus lactis or Lactobacillus johnsonii cell.

### Claim 27 (depends on 26)

The host cell according to claim 26 wherein the host cell is a Lactococcus lactis cell selected from the group consisting of L. lactis F110676 and L. lactis UKLc10.

### Claim 28 (depends on 26)

The host cell according to claim 26 wherein the host cell is a Lactobacillus johnsonii cell selected from the group consisting of L. johnsonii F19785 and L. johnsonii FI10744.

### Claim 29 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the fusion polypeptide further comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 322 to 375 of SEQ ID NO: 1.

### Claim 30 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the fusion polypeptide further comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 135 to 321 of SEQ ID NO: 1.

### Claim 31 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the polypeptide having endolysin activity comprises the amino acid sequence of SEQ ID NO: 1 and the fusion polypeptide is capable of binding specifically to cells of Clostridium perfringens and/or lysing cells of Clostridium perfringens.

### Claim 32 (depends on 13)

The expression vector according to claim 13, wherein the polypeptide further comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 322 to 375 of SEQ ID NO: 1.

### Claim 33 (depends on 13)

The expression vector according to claim 13, wherein the polypeptide further comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 135 to 321 of SEQ ID NO: 1.

### Claim 34 (depends on 13)

The expression vector according to claim 13, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and is capable of binding specifically to cells of Clostridium perfringens and/or lysing cells of Clostridium perfringens.

### Claim 35 (depends on 22)

The host cell according to claim 22, wherein the polypeptide further comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 322 to 375 of SEQ ID NO: 1.

### Claim 36 (depends on 22)

The host cell according to claim 22, wherein the polypeptide further comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of amino acids 135 to 321 of SEQ ID NO: 1.

### Claim 37 (depends on 22)

The host cell according to claim 22, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and is capable of binding specifically to cells of Clostridium perfringens and/or lysing cells of Clostridium perfringens.