Process to produce fibrillar proteins and method of treating using fibrillar proteins

A fibrous protein, protein technology, applied in the preparation method of peptide, peptide/protein composition, animal/human protein, etc., can solve the problem of long time and so on

Active Publication Date: 2014-12-17
ACAD SINIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods usually require high concentration of protein, vigorous shaking, the assistance of fibril seeds, and the time is very long, and sometimes even need to incubate at a specific temperature for about a month

Method used

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  • Process to produce fibrillar proteins and method of treating using fibrillar proteins
  • Process to produce fibrillar proteins and method of treating using fibrillar proteins
  • Process to produce fibrillar proteins and method of treating using fibrillar proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Materials and methods

[0054] Material: Phospho-Ser 473 Akt antibody was purchased from Cell Signaling Technology. Zwittergent 3-14 was purchased from Calbiochem. Fibronectin (Fibronectin, FN), anti-actin antibody (anti-actin antibody), anti-integrin α5β1 polyclonal antibody (anti-integrin α5β1 polyclonal antibody) (function inhibitory antibody), horseradish peroxidase-conjugated antibody Mouse IgG secondary antibodies (horseradish peroxidase-coupled anti-mouse IgG secondary antibo dies), horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (horseradish peroxidase-coupled anti-rabbit IgG secondary antibodies) and MTT test group were purchased from Chemicon International Corporation. Bovine serum albumin (BSA) was purchased from Bio Basic. Thioflavin T (Thioflavin, ThT) and sodium dodecyl sulfate (SDS) were purchased from Sigma.

[0055] Expression and purification of recombinant proteins VP1 and VP3. VP1 and VP3 are the capsid protein components of fo...

Embodiment 2

[0088] Materials and methods

[0089] Material: Phosphorus-Try 576 / 577 FAK(phospho-Try 576 / 577 FAK), phosphorus-Ser 473 Akt(phospho-Ser 473 Akt) and phosphorus-Ser 9 GSK-3β(phospho-Ser 9 GSK-3β) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Phosphorus-Tyr 397 FAK(phospho--Tyr 397 FAK) antibodies were purchased from Biosource (Camerillo, California, USA). Zwittergent 3-14 was purchased from Calbiochem (San Diego, California, USA). Integrin α5β1 protein, anti-β-actin antibody, anti-integrin α5 antibody, anti-integrin α5β1 antibody (anti-integrin α5β1 antibody) ) (Functional inhibitory antibody), horseradish peroxidase-coupled anti-mouse IgG secondary antibodies, horseradish peroxidase-coupled anti-mouse IgG secondary antibodies, horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (horseradish peroxidase -coupled anti-rabbit IgG secondary antibodies) and the MTT test set were purchased from Chemicon (Timekla, California). Anti-BSA an...

Embodiment 3

[0110] Previous experiments found that the use of Superdex-75 in the presence of 8M urea for bovine serum albumin can induce unfolded bovine serum albumin to have a fibril form; the recombinant protein VP1 also has the same situation, with fibril formation. This result can be determined by the increase in ThT ( Figure 8 ) And cytotoxic ( Picture 9 ) And confirmed. The use concentration of urea is not limited to 8M, and other molar ratios can also promote protein unfolding to the same or lesser extent.

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PUM

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Abstract

A method for changing a globular protein structure into a fibrillar protein structure. The method comprising the steps of providing a globular protein, forming a solution containing the globular protein, adding a detergent to the solution containing the globular protein, applying the solution to a molecular sizing column with a pore size of at least 70 kDa and eluting with a solution containing detergent. A method for changing an unfolded protein structure into a fibrillar protein structure. The method comprising the steps of providing a globular protein, forming a solution containing the globular protein, adding a urea to the solution to unfold the globular protein, applying the solution to a molecular sizing column and eluting with a solution containing detergent. A method for treating cancer comprising the steps of providing a protein, changing the protein into a fibrillar structure, and administering a therapeutically effective amount of the fibrillar structure protein to a patient in need thereof. A method for producing a vaccine adjuvant or antigen adjuvant comprising the steps of providing a protein, and changing the protein into a fibrillar structure.

Description

Technical field [0001] The present invention relates to a method for folding unfolded native protein (native protein) into a fibrillar structure, and the present invention also finds that the use of fibrillar structure protein can induce cell apoptosis and act as a vaccine adjuvant (adjuvant). ). [0002] This application requires U.S. Provisional Application No. 12 / 082,634 filed on April 11, 2008, British Application No. 0809919.4 filed on May 30, 2008, and U.S. Provisional Application No. filed on March 13, 2008 The priority of No. 61 / 036,432, the entire disclosure of which is hereby incorporated by reference. Background technique [0003] Previous studies have been found in glycation (Bouma et al. published in J Bio Chem 278(43):41810-41819:2003) and high temperature culture (Sagis et al. published in Langmuir 20(3):924-927:2004) , Or ultrasonic shock (Stathopulos et al. published in Protein Sci 13(11): 3017-3027: 2004), some proteins will form fibrous structures. However, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/107C07K1/14C07K14/47A61K38/17A61P35/00A61K38/16
CPCC12N2770/32151A61K38/162C12N2770/32122C07K14/78C07K1/1136A61K38/38A61K38/39C07K14/765A61P35/00A61P37/04
Inventor 杨淑美梁启铭陈彦伯黄俊勇
Owner ACAD SINIC
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