Multimeric antimicrobial peptide complex which is displayed on cell surface

Patent metadata

Field	Value
Publication number	US-10406204-B2
Application number	US-201514846637-A
Country	US
Kind code	B2
Filing date	Sep 4, 2015
Priority date	Dec 6, 2010
Publication date	Sep 10, 2019
Grant date	Sep 10, 2019

Abstract

The present invention provides an antimicrobial peptide polymer comprising at least one monomer which is digested by pepsin, a multimeric antimicrobial peptide complex comprising the polymer and a cell surface anchoring motif linked to the polymer, an antimicrobial microorganism displaying the multimeric antimicrobial peptide complex, an antimicrobial composition comprising the same, a method of treating an infectious disease caused by bacteria, yeast or fungi by administering the antimicrobial composition, and a method for producing the antimicrobial microorganism. According to the invention, living microorganisms displaying an antimicrobial peptide on the cell surface thereof may be administered in vivo without having to lyse the microbial cell and isolate and purify the antimicrobial peptide, so that the antimicrobial peptide exhibits antimicrobial activity. Thus, the antimicrobial peptide may be produced at significantly reduced costs so that it may have widespread use.

First claim

The invention claimed is:

1. A method for producing an antimicrobial transformant displaying a multimeric antimicrobial peptide complex on the cell surface thereof, comprising
(a) preparing a recombinant vector comprising a polynucleotide encoding the multimeric antimicrobial peptide complex comprising a cell surface anchoring motif linked to the antimicrobial peptide via an amino acid linker, wherein the complex is represented by the following formula 1 or 2:
(1) a cell surface anchoring motif—[an amino acid linker that can be digested randomly by pepsin—an antimicrobial peptide] n ,
(2) a cell surface anchoring motif—[an antimicrobial peptide—an amino acid linker that can be digested randomly by pepsin] n ,

wherein n is an integer greater than 1; and
wherein the antimicrobial peptide does not comprise leucine, phenylalanine, and tyrosine;
both amino acid terminuses of the linker are an amino acid selected from the group consisting of leucine, phenylalanine and tyrosine;
the peptide linkage formed between the end of the linker and the terminus of the antimicrobial peptide, and the peptide linkage formed between the end of the linker and the terminus of the cell surface anchoring motif can be cleaved by the action of the extracellular digestive enzyme pepsin; and
the cell surface anchoring motif is selected from the group consisting of outer membrane proteins, lipoproteins, autotransporters, and S-layer of a surface appendage;
(b) introducing the recombinant vector into a host cell to obtain a transformant; and
(c) culturing the transformant to induce the expression of the multimeric antimicrobial peptide complex, wherein the expressed multimeric antimicrobial peptide complex is displayed and anchored to the surface of the antimicrobial transformant.

2. The method for producing the antimicrobial transformant of claim 1 , wherein the antimicrobial peptide has any one of amino acid sequences represented by SEQ ID NOS: 9 to 24.

3. The method for producing the antimicrobial transformant of claim 1 , wherein the cell surface anchoring motif is linked to the N-terminus of the polymer.

4. The method for producing the antimicrobial transformant of claim 1 , wherein the cell surface anchoring motif is an outer membrane protein.

5. The method for producing the antimicrobial transformant of claim 4 , wherein the outer membrane protein is selected from the group consisting of an E. coli outer membrane protein OmpA, an E. coli outer membrane protein OmpA linked to the leader sequence of E. coli lipoprotein, an E. coli outer membrane protein OmpS, an E. coli outer membrane protein LamB, an E. coli outer membrane protein PhoE, an E. coli outer membrane protein OmpC, an E. coli outer membrane protein FadL, a Salmonella outer membrane protein OmpC, and a Pseudomonas outer membrane protein OprF.

6. The method for producing the antimicrobial transformant of claim 1 , wherein the cell surface anchoring motif is an E. coli outer membrane protein OmpA linked to the leader sequence of E. coli lipoprotein which comprises the sequence of SEQ ID NO: 8.

7. The method for producing the antimicrobial transformant of claim 1 , wherein the antimicrobial peptide comprises the sequence of SEQ ID NO: 9, the amino acid linker digested by pepsin is leucine, and the cell surface anchoring motif comprises the sequence of SEQ ID NO: 8.

Assignees

• Korea Advanced Inst Sci & Tech
• Intelligent Synthetic Biology Ct

Inventors

• Kim Sun-Chang
• Shin Ju Ri
• Lim Ki Jung
• Kim Da Jung
• Lee Young Woong
• Jang Su A
• Sung Bong Hyun

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• C07K14/4723

  Cationic antimicrobial peptides, e.g. defensins · CPC title

• C07K14/255

  Salmonella (G) · CPC title

• C07K14/21

  from Pseudomonadaceae (F) · CPC title

• A61K47/65

  Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers · CPC title