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Method of monitoring cellular trafficking of peptides

A cell and host cell technology, applied in the detection of programmed cell death, biochemical equipment and methods, biological testing, etc., can solve the problems of inability to select peptides, and cannot provide verification of cell internalization or delivery, etc., to reduce hydrophobicity Effect

Inactive Publication Date: 2016-06-08
PHYLOGICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although phage display screening techniques are widely and successfully used to discover new CPPs, existing screening methods cannot necessarily select peptides based on properties beyond cellular uptake and cannot provide validation of cellular internalization or delivery

Method used

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  • Method of monitoring cellular trafficking of peptides
  • Method of monitoring cellular trafficking of peptides
  • Method of monitoring cellular trafficking of peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0338] Generation of candidate peptide moieties

[0339] This example demonstrates the use of nucleic acids encoding candidate peptides to generate candidate peptide moieties, eg, peptide libraries such as bacteriophage display libraries or other peptide display scaffolds.

[0340] High diversity mixtures of nucleic acids encoding candidate peptides are generated from the coding and non-coding regions of bacterial genomes and eukaryotes with compact genomes, as essentially described in U.S. Patent No. 7,270,969, and as described below, the selected source genome can vary , and the vector used for expression of the peptide encoded by the nucleic acid described in the Examples below may vary. The contents of US Patent No. 7,270,969 are incorporated herein by reference in their entirety.

[0341] Briefly, nucleic acids were isolated from the following bacterial and archaeal species:

[0342]

[0343]

[0344] Nucleic acid fragments were generated separately from these gen...

Embodiment 2

[0353] Production of non-biotinylated fractions using the expression vector pNp3

[0354] This example demonstrates the production of non-biotinylated components using the expression vector pNp3 or derivatives thereof to obtain filamentous bacteriophage displaying non-biotinylated components.

[0355] The designated vector construct, pNp3, is an M13 vector comprising an encoded fusion protein containing a hexahistidine (6His) tag, a hemagglutinin (HA) tag, a biotin ligase substrate domain, and the M13pIII coat protein. Vector pNp3 was modified to express a fusion protein comprising a candidate peptide moiety fused in-frame with a 15-amino acid biotin ligase substrate domain having the amino acid sequence shown in SEQ ID NO: 4, as shown in Figure 1a, 1b and 1c are shown. Fusion proteins generated using pNp3 were subsequently displayed on scaffolds containing filamentous bacteriophage M13.

[0356] Figure 1a shows the pIII fusion protein encoded by the pNp3 derivative vector P...

Embodiment 3

[0376] Production of non-biotinylated fractions using the expression vector pNp8

[0377] This example demonstrates the production of non-biotinylated components using the expression vector pNp8 or derivatives thereof to obtain filamentous bacteriophage displaying non-biotinylated components.

[0378] The designated vector construct, pNp8, is an M13 vector comprising a fusion protein encoding a hexahistidine (10His) tag, a hemagglutinin (HA) tag, a biotin ligase substrate domain, and the M13pVIII coat protein. Vector pNp8 was modified to express a fusion protein comprising a candidate peptide moiety fused in-frame to a 15-amino acid biotin ligase substrate domain having the amino acid sequence shown in SEQ ID NO: 4, as shown in Figures 4a and 4b. Fusion proteins generated using pNp8 were subsequently displayed on scaffolds containing filamentous bacteriophage M13.

[0379] Figure 4a shows the pVIII fusion protein encoded by the pNp8 derivative vector PelB-Avitag-pVIII, which ...

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Abstract

ABSTRACT This disclosure provides a method of isolating peptides having cell-penetrating function, wherein the peptides are detected as biotinylated molecules only following their translocation through the cell membrane. The disclosure also provides methods for validating the cell-penetrating function of the peptides, or that may be employed in their own right to isolate such peptides, wherein the peptides are detectable by virtue of their ability to transport a detectable cargo into the cytoplasm, such as a cargo toxin or a fragment of a green fluorescent protein (GFP) that is required for complementation of a functional GFP. The disclosure also provides non-canonical peptides having cell-penetrating function that differ structurally from known CPPs such as TAT, VP22, transportan and penetratin, and that are capable of translocating cell membranes and escaping the endosome. The disclosed peptides have utility in transporting cargo therapeutics and diagnostics into cells.

Description

[0001] related application [0002] This application claims the priority of Australian Patent Application No. 2013902347 filed on June 26, 2013 and Australian Patent Application No. 2013903038 filed on August 13, 2013 and Australian Patent Application No. 2014901714 filed on May 9, 2014 in accordance with the Convention, The contents of said documents are each incorporated herein by reference in their entirety. technical field [0003] The present invention relates generally to the field of pharmaceutical sciences and, in particular, to the targeting of molecules such as therapeutic compounds and peptides to organs and / or tissues and / or cellular and / or subcellular locations. Background of the invention [0004] Many biologically active compounds require intracellular delivery, either within the cytoplasm, nucleus or other organelles, to exert their therapeutic effects. Selective delivery to specific organs, tissues, cellular or subcellular locations is highly desirable to a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N33/52C12N15/62
CPCG01N33/52C12Q1/25G01N33/68G01N33/5035G01N2333/9015G01N2440/32G01N2500/10
Inventor R·霍普金斯K·霍夫曼T·海因里希P·坎宁安P·沃特N·米勒什
Owner PHYLOGICA
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