Di-enzymatic chimeric endolysin

Patent metadata

Field	Value
Publication number	US-10975365-B2
Application number	US-201916670194-A
Country	US
Kind code	B2
Filing date	Oct 31, 2019
Priority date	Nov 5, 2018
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

A di-enzymatic chimeric endolysin includes a primary enzymatic active domain including a primary protein sequence and that cleaves a glycosidic, peptide, or amide bond; a secondary enzymatic active domain disposed at a C-terminus end of the di-enzymatic chimeric endolysin and including a secondary protein sequence that, in combination with the primary enzymatic active domain, synergistically cleaves glycosidic, peptide, or amide bonds in a peptidoglycan; a cell wall binding domain including a recognition sequence that is sequentially interposed between the primary protein sequence and the secondary protein sequence and that binds to a cell wall; and a tertiary structure such that the primary enzymatic active domain faces and opposes the secondary enzymatic active domain in the di-enzymatic chimeric endolysin for synergistic cleavage of the peptidoglycan.

First claim

What is claimed is:

1. A di-enzymatic chimeric endolysin comprising:
a primary enzymatic active domain disposed at an N-terminus end of the di-enzymatic chimeric endolysin, the primary enzymatic active domain comprising a primary protein sequence and that cleaves a glycosidic bond, a peptide bond, or an amide bond of a peptidoglycan in a cell wall of a cell;
a secondary enzymatic active domain disposed at a C-terminus end of the di-enzymatic chimeric endolysin, the secondary enzymatic active domain comprising a secondary protein sequence and that, in combination with the primary enzymatic active domain, synergistically cleaves glycosidic bonds, peptide bonds, or amide bonds in the peptidoglycan in the cell wall;
a cell wall binding domain:
comprising a recognition sequence;
chemically attached to the primary protein sequence and the secondary protein sequence,
sequentially interposed between the primary protein sequence and the secondary protein sequence, and
that binds to a cell wall; and

a tertiary structure formed by folding of the primary protein sequence and the secondary protein sequence such that the primary enzymatic active domain faces and opposes the secondary enzymatic active domain in the di-enzymatic chimeric endolysin for synergistic cleavage of the peptidoglycan in the cell wall.

2. The di-enzymatic chimeric endolysin of claim 1 , further comprising a first linker interposed between the primary protein sequence and the recognition sequence.

3. The di-enzymatic chimeric endolysin of claim 2 , wherein the first linker comprises:

(Sequence ID No. 4)

TGDGKNPSVGTGNATVSASSE

or

(Sequence ID No. 5)

TGDGKNPSVGTGNATVSASSECT.

4. The di-enzymatic chimeric endolysin of claim 1 , further comprising a second linker interposed between the secondary protein sequence and the recognition sequence.

5. The di-enzymatic chimeric endolysin of claim 4 , wherein the second linker comprises

(Sequence ID No. 6)

QTNPNPDKPTVKSPGQNDLGS

or

(Sequence ID No. 7)

LQQTNPNPDKPTVKSPGQNDLGS.

6. The di-enzymatic chimeric endolysin of claim 1 , wherein the primary protein sequence comprises

(Sequence ID No. 1)

MSKKYTQQQYEKYLAQPANNTFGLSPQQVADWFMGQAGARPVINSYGVNAS

NLVSTYIPKMQEYGVSYTLFLMYTVFEGGGAGNWINHYMYDTGSNGLECLE

HDLQYIHGVWETYFPPALSAPECYPATEDNAGALDRFYQSLPGRTWGDVMI

PSTMAGNAWVWAYNYCVNNQGAAPLVYFGNPYDSQIDSLLAMGADPFTGGS

I,

(Sequence ID No. 2)

MVKKNDLFVDVSSHNGYDITGILEQMGTTNTIIKISESTTYLNPCLSAQVE

QSNPIGFYHFARFGGDVAEAEREAQFFLDNVPMQVKYLVLDYEDDPSGDAQ

ANTNACLRFMQMIADAGYKPIYYSYKPFTHDNVDYQQILAQFPNSLWIAGY

GLNDGTANFEYFPSMDGIRWWQYSSNPFDKNIVLLDD,

or

(Sequence ID No. 3)

MTTVNEALNNVRAQVGSGVSVGNGECYALASWYERMISPDATVGLGAGVGW

VSGAIGDTISAKNIGSSYNWQANGWTVSTSGPFKAGQIVTLGATPGNPYGH

VVIVEAVDGDRLTILEQNYGGKRYPVRNYYSAASYRQQVVHYIT.

7. The di-enzymatic chimeric endolysin of claim 1 , wherein the secondary protein sequence comprises

(Sequence ID No. 8)

GSDRVAANLANAQAQVGKYIGDGQCYAWVGWWSARVCGYSISYSTGDPMLP

LIGDGMNAHSIHLGWDWSIANTGIVNYPVGTVGRKEDLRVGAIWCATAFSG

APFYTGQYGHTGIIESWSDTTVTVLEQNILGSPVIRSTYDLNTFLSTLTGL

ITFK

or

(Sequence ID No. 9)

MVKKNDLFVDVSSHNGYDITGILEQMGTTNTIIKISESTTYLNPCLSAQVE

QSNPIGFYHFARFGGDVAEAEREAQFFLDNVPMQVKYLVLDYEDDPSGDAQ

ANTNACLRFMQMIADAGYKPIYYSYKPFTHDNVDYQQILAQFPNSLWIAGY

GLNDGTANFEYFPSMDGIRWWQYSSNPFDKNIVLLDDEEDDKPKTAGTWKQ

DSKGWWFRRNNGSFPY.

8. The di-enzymatic chimeric endolysin of claim 1 , wherein the recognition sequence comprises

(Sequence ID No. 10)

ANREKLKKALTDLFNNNLEHLSGEFYGNQVLNAMKYGTILKCDLTDDGLNA

ILQLIADVNL,

Assignees

• Government Of The Us Secretary Of Commerce

Inventors

• Kelman Zvi
• Nelson Daniel Craig
• Shang Xiaoran

Classifications

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12N9/50

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title

• C12N1/06

  Lysis of microorganisms · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title