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Compositions and methods for protein display on the surface of bacteria and vesicles derived therefrom and uses thereof

A protein and application technology, applied in bacteria, combinatorial chemistry, inactive components of polymer compounds, etc., can solve the problem of not carrying signal peptides

Active Publication Date: 2019-12-17
CORNELL RES FOUNDATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pathway by which ClyA manages to cross the bacterial IM and assemble in the OMV remains a mystery because it does not carry a conventional signal peptide (del Castillo et al., “The Escherichia coli K-12 SheA Gene Encodes a 34-kDa Secreted Haemolysin (Escherichia coli K-12 SheA gene encodes a 34-kDa secreted hemolysin), Mol Microbiol 25:107-15 (1997)), and is not processed at the N-terminus (Ludwig et al., "Analysis of the SlyA-Controlled Expression ,Subcellular Localization and Pore-Forming Activity of a 34 kDa Haemolysin (ClyA) from Escherichia coli K-12 (SlyA-controlled expression, subcellular localization and pore-forming activity analysis of Escherichia coli K-1234 kDa hemolysin (ClyA))", Mol Microbiol 31:557-67 (1999))

Method used

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  • Compositions and methods for protein display on the surface of bacteria and vesicles derived therefrom and uses thereof
  • Compositions and methods for protein display on the surface of bacteria and vesicles derived therefrom and uses thereof
  • Compositions and methods for protein display on the surface of bacteria and vesicles derived therefrom and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Example 1 - Bacterial Strains, Plasmids and Growth Conditions

[0193] The bacterial strains and plasmids used in these examples are described in Table 1.

[0194] Table 1. Bacterial Strains and Plasmids

[0195]

[0196]

[0197]

[0198]

[0199]Using DHA as a donor, the dsbA::Kan allele was introduced into JC8031 via P1vir transduction to prepare cell strain JCA. The PCR-amplified clyA gene was ligated with pBAD18-Cm between SacI and XbaI sites to construct plasmid pClyA. The gene encoding gfpmut2 was inserted between the XbaI and HindIII sites respectively (Crameri et al., "Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling", Nat Biotechnol 14:315-9 (1996) and DeLisa et al., "Genetic Analysis of the Twin Arginine Translocator Secretion Pathway inBacteria (the genetic analysis of the Twin Arginine Translocator Secretion Pathway in bacteria)", J Biol Chem 277:29825-31 (2002), described The literature is hereby incorporated by ...

Embodiment 2

[0200] Example 2 - Cell Culture

[0201] Human epithelial cervical carcinoma (HeLa) cells were obtained from the American Type Culture Collection (ATCC #CCL-2) and cultured in Dulbecco's modified Eagles supplemented with 10% NuSerum and 1% penicillin / streptomycin. Grow in minimal medium (DMEM). Maintain cells at 37°C, 95% air, 5% CO 2 in a humidified atmosphere. For fluorescence microscopy experiments, cells were grown on 12-mm round glass coverslips for 2 days prior to the experiment.

Embodiment 3

[0202] Example 3 - Subcellular Fractionation

[0203] By cold osmotic shock procedure (cold osmotic shock procedure) (Kim et al., "Twin-ArginineTranslocation of Active Human Tissue Plasminogen Activator in Escherichiacoli (Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli) bit)", Applied and Environmental Microbiology 71:8451-8459 (2005), which is incorporated herein by reference in its entirety), the cytoplasmic and periplasmic fractions were prepared from cells expressing the fusion protein, and the soluble fraction was removed The remaining precipitate was then collected as the insoluble fraction.

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Abstract

The present invention relates to compositions and methods for displaying proteins and polypeptides on the surface of cells and vesicles. Also disclosed herein are methods and compositions for the delivery of drugs and vaccines utilizing the cell surface display systems of the present invention.

Description

[0001] This application is a divisional application of the following applications: filing date: May 21, 2008; application number: 200880024976.5 (PCT / US2008 / 064376); title of invention: same as above. [0002] This application claims the benefit of US Provisional Patent Application Serial No. 60 / 939,506 filed May 22,2007. [0003] The subject matter of this application was made with support from the United States Government under the National Institutes of Health under grant number NIBIB R21EB005669. The U.S. government has certain rights. [0004] field of invention [0005] The present invention relates to compositions and methods for the display of proteins and polypeptides on the surface of cells and vesicles. [0006] Background of the invention [0007] Protein translocation is a highly conserved process essential to all life. Extracellular secretion of virulence factors is a strategy used by invading bacteria to establish a colony niche, communicate with host cells, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N1/21C40B30/04A61K47/42A61K39/39A61K39/385
CPCA61K39/385A61K39/39A61K47/42A61K2039/55544A61K2039/6037C12N15/1037C40B30/04
Inventor M.德利萨J.金D.A.普特曼A.M.杜迪
Owner CORNELL RES FOUNDATION INC
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