Reversibly assembled and disassembled Strep-tag polypeptide-labeled biomolecule derivatization matrix and application thereof

A biomolecular and derivatization technology, applied in the fields of biology, chemistry and material science, can solve the problems of cumbersome probe preparation process, complicated operation process, lack of specificity, etc., and achieve simple operation preparation and use process, good repeatability, The effect of less cell damage

Inactive Publication Date: 2015-03-25
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are many reports on the capture and release of circulating tumor cells, but there are often problems such as lack of specificity, cumbersome probe preparation process, complicated operation process, and greater damage to cells during the capture and release process. The non-destructive isolation and in vitro culture of cells are still rarely reported

Method used

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  • Reversibly assembled and disassembled Strep-tag polypeptide-labeled biomolecule derivatization matrix and application thereof
  • Reversibly assembled and disassembled Strep-tag polypeptide-labeled biomolecule derivatization matrix and application thereof
  • Reversibly assembled and disassembled Strep-tag polypeptide-labeled biomolecule derivatization matrix and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034](1) Disperse 1mg of goat anti-mouse IgG (secondary antibody) in 1mL of 0.1M PBS with pH 6.4, mix with 20mM sodium periodate solution 1:1 (v / v), and incubate at room temperature in the dark For 1 h, wash with 1 mL of 0.1 M PBS with pH 6.4 to remove excess sodium periodate and disperse in 1 mL of 0.1 M PBS with pH 6.4 to obtain the oxidized secondary antibody.

[0035] (2) Mix Strep-tag II polypeptide at a concentration of 0.3 mg / mL with oxidized secondary antibody (200 μL, 1 mg / mL) at a molar ratio of 20:1 (polypeptide: antibody), incubate at room temperature for 30 min, and ultrafilter Wash 3 times to remove excess peptide and disperse in 200 μL 0.1M PBS, pH 7.2 to obtain peptide-labeled secondary antibody.

[0036] (3) Mix 100 μL of commercially available resin balls loaded with Strep-Tactin at a concentration of 5 mg / mL with 100 μL of Strep-tag polypeptide-labeled secondary antibody (1 mg / mL), incubate at room temperature for 30 min, wash 3 times and disperse in 100 μL...

Embodiment 2

[0039] (1) Disperse 1mg of goat anti-mouse IgG (secondary antibody) in 1mL of 0.1M PBS with pH 6.4, mix with 20mM sodium periodate solution 1:1 (v / v), and incubate at room temperature in the dark For 1 h, wash with 1 mL of 0.1 M PBS with pH 6.4 to remove excess sodium periodate and disperse in 1 mL of 0.1 M PBS with pH 6.4 to obtain the oxidized secondary antibody.

[0040] (2) Mix the Strep-tag II polypeptide with a concentration of 0.3mg / mL and the oxidized secondary antibody (200μL, 1mg / mL) at a molar ratio of 3:1, incubate at room temperature for 30min, and wash with ultrafiltration for 3 times to remove excess polypeptide And dispersed in 200 μL of 0.1M PBS with pH7.2 to obtain the peptide-labeled secondary antibody.

[0041] (3) Take 10 μL of commercially available magnetic beads loaded with Strep-Tactin at a concentration of 50 mg / mL, wash 2 times with 100 μL buffer W (100 mM Tris / HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), and then disperse in 200 μL of peptide-labeled second...

Embodiment 3

[0044] (1) Disperse 1mg of goat anti-mouse IgG (secondary antibody) in 1mL of 0.1M PBS with pH 6.4, mix with 20mM sodium periodate solution 1:1 (v / v), and incubate at room temperature in the dark For 1 h, wash with 1 mL of 0.1 M PBS with pH 6.4 to remove excess sodium periodate and disperse in 1 mL of 0.1 M PBS with pH 6.4 to obtain the oxidized secondary antibody.

[0045] (2) Mix the Strep-tag II polypeptide with a concentration of 0.3mg / mL and the oxidized secondary antibody (200μL, 1mg / mL) at a molar ratio of 3:1, incubate at room temperature for 30min, and wash with ultrafiltration for 3 times to remove excess polypeptide And dispersed in 200 μL of 0.1M PBS with pH7.2 to obtain polypeptide-labeled antibody.

[0046] (3) Take 10 μL of magnetic beads loaded with Strep-Tactin at a concentration of 50 mg / mL, wash 2 times with 100 μL buffer W (100 mM Tris / HCl pH 8.0, 150 mM NaCl, 1 mM EDTA), disperse in 200 μL of peptide-labeled antibody, and incubate for 30 min After that, w...

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Abstract

The invention discloses a reversibly assembled and disassembled Strep-tag polypeptide-labeled biomolecule derivatization matrix and an application thereof. Polypeptide can be linked on the surfaces of different matrixes on the basis of the characteristic that Strep-tag polypeptide can be bound to a matrix loaded with Strep-Tactin protein. Chemical derivatization is carried out on biological targeting molecules so as to form a biomolecule-Strep-tag conjugate; by virtue of specific recognition effects of the Strep-tag and the Strep-Tactin, the biomolecule-Strep-tag conjugate is immobilized on the surface of the matrix. After biotin is added, the linkage between the biomolecule and the matrix is destroyed due to the competitive binding between the biotin and the Strep-Tactin, so that reversible trapping and releasing on a to-be-detected object are achieved. The matrix is simple and rapid in preparing and using processes, good in repeatability, and is widely applicable to such fields of tumor marker detection, cell trapping and releasing, and the like.

Description

[0001] technical field [0002] The invention belongs to the fields of biology, chemistry and material science, and relates to a reversibly assembled and decomposed Strep-tag polypeptide-marked biomolecule derivatized matrix and its application. Background technique [0003] The Strep-tag purification system is a protein purification system based on the highly selective and easily controllable interaction between the Strep-tag polypeptide and the specially designed Strep-Tactin (a variant of streptavidin). The principle of its technology development is Binding reaction between biotin and streptavidin. Strep-tag polypeptides include Strep-tag I and Strep-tag II, which have higher binding affinity to Strep-Tactin than to streptavidin. During affinity purification, the tagged protein binds to the immobilized Strep-Tactin. After a short washing step, the purified recombinant protein is gently eluted by adding the specific competitor biotin in the same buffer. The system is si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30B01D15/38C12N5/00
CPCB01J20/24B01D15/38B01J20/30C12N5/00
Inventor 黄卫华陆宁宁谢敏程世博
Owner WUHAN UNIV
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