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Bacterial surface display and screening of thioether-bridge-containing peptides

A thioether, fusion peptide technology, applied in the direction of peptides, fusion peptides, chemical instruments and methods, etc., can solve problems such as difficulty in introducing synthetic peptides

Inactive Publication Date: 2013-05-01
LANTHIO PEP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, it is still difficult to efficiently introduce such structures into synthetic peptides, especially large ones

Method used

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  • Bacterial surface display and screening of thioether-bridge-containing peptides
  • Bacterial surface display and screening of thioether-bridge-containing peptides
  • Bacterial surface display and screening of thioether-bridge-containing peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Expression vectors for cell surface display of peptides containing thioether and dehydrogenation residues on Lactococcus lactis

[0059] Target : This example is about the construction of an expression vector for displaying a thioether-containing peptide on the surface of Lactococcus lactis. The Lactococcus lactis host organism provides the machinery for the introduction of thioether-linked nisin biosynthesis and export in the desired peptide and its export. The peptide is translationally fused to the LPXTG cell wall anchor motif, eg, Lactococcus lactis PrtP protease. This anchoring mechanism requires the processing of sortase for the covalent anchoring of peptides to the peptidoglycan of the bacterial cell wall. In this way, the peptide and the encoding DNA are linked, allowing selection / screening of post-translationally modified peptides with desired properties. Nisin will be used as a model peptide for the development and validation of the display syst...

Embodiment 2

[0063] Example 2: Antibodies against the leader peptide demonstrate attachment of the pronisin to the cell surface location of the cell wall of the fusion protein TB5.

[0064] Target : Evaluation of TB5 display on the cell surface of Lactococcus lactis by whole-cell ELISA using the anti-nisin leader peptide

[0065] Materials and methods

[0066] L. lactis NZ9000 (pTB5) and L. lactis NZ9000 (pIL3BTC / pTB5) cells were cultured in the presence or absence of nisin to induce TB5. After preparation, the produced cells were collected by centrifugation and washed three times with pH 7.4 phosphate-buffered saline (PBS). Equal numbers of TB5-displaying cells were incubated with 1000-fold diluted rabbit anti-nisin lead antibody solution in PBS containing 0.5% BSA in a final volume of 1 ml for 1 hr at room temperature with agitation. After three washes with PBS, the displayed TB5 was visualized by incubation with goat anti-rabbit IgG conjugated to alkaline phosphatase (1:10000) and...

Embodiment 3

[0069] Example 3: Prenisin targeting fusion proteins have been intracellularly modified with NisB and NisC prior to cell surface display. Modification by NisB- and NisC-modified prenisin-anchored fusion proteins was demonstrated by antibacterial activity against overlapping cells after leader peptide cleavage.

[0070] Target : Example 2 shows that production of TB5 results in the display of pro-nisin on the cell surface of Lactococcus lactis. In this example, nisin modification by NisB and NisC was assessed with an overlay of a nisin-sensitive Lactococcus lactis strain. Growth inhibition of this strain indicated that NisB and NisC correctly modified TB5 and formed at least loops A, B and C.

[0071] Materials and methods

[0072] Overlay of plaques with well-washed SDS-PAA gels or pTB5-producing L. lactis NZ9000 cells with 200-fold dilutions of L. lactis MG1363 or NZ9000 strains in 0.5% top agar containing 0.1 mg / ml trypsin GM17 agar plates. Trypsin is required for cl...

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Abstract

The invention relates to bacterial cell surface display of post-translationally modified heterologous proteins. Provided is an isolated nucleic acid construct encoding a proteinaceous substance comprising, from the N-terminus to the C-terminus, at least (a) an N-terminal a lantibiotic leader sequence; (b) an amino acid sequence of interest to be post-translationally modified to a dehydroresidue- or thioether-bridge containing polypeptide; (c) a hydrophilic cell-wall spanning domain; (d) a sortase recognition motif; (e) a hydrophobic membrane spanning domain and (f) a C-terminal charged membrane anchoring domain. Also provided is a Gram-positive host cell expressing the construct, as well as a library of host cells.

Description

technical field [0001] The present invention relates to the field of protein engineering and screening of therapeutically relevant peptides. More specifically, it relates to the cell surface display of post-translationally modified heterologous proteins. Heterologous display of proteins or peptides on the surface of microorganisms, such as bacteria, is a useful research tool and has been associated with a wide range of interesting applications. Linking protein or peptide function to an encoding gene enables screening and / or optimization of peptides with desired properties from large combinatorial libraries. A variety of display formats have been developed, including ribosomal display, phage display, bacterial surface display, and yeast display. Phage display is perhaps the best known system. Background technique [0002] One of the most interesting applications of cell surface display is the selection of high-affinity ligands for a therapeutic target molecule of interest ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/10
CPCC12N15/625C07K2319/50C12N15/1037C07K2319/035G01N33/6803
Inventor 芝贝·博斯姆
Owner LANTHIO PEP
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