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Polypeptide for protein surface immobilization and application thereof

A fusion protein and silica technology, applied in the protein field, can solve the problems of damage to the protein, increase the time and cost of the immobilization process, and not site-specific binding, etc., to achieve the effect of increasing affinity, improving possibility, and optimizing function

Active Publication Date: 2020-04-03
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, this method has the following disadvantages: (1) requires the use of hazardous chemicals, (2) requires an additional reaction step, which increases the time and cost of the fixation process, and (3) uncontrolled fixation orientation may damage the protein function of
For example, the binding site of an antibody may be blocked during chemical cross-linking because the binding is not site-specific

Method used

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  • Polypeptide for protein surface immobilization and application thereof
  • Polypeptide for protein surface immobilization and application thereof
  • Polypeptide for protein surface immobilization and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 2

[0055] Example 1 Preparation of silica-bound peptide anti-CEA nanobody fusion protein

[0056] 1. Construction of Silica-Conjugated Peptide Anti-CEA Nanobody Fusion Protein

[0057] The silica-binding peptide was reconstituted into an anti-CEA heavy chain antibody (clone no. 2D5, 11C12) isolated from an immunized llama following the procedure described in WO 94 / 25591. For information about the 2D5 heavy chain antibody, please refer to Chinese patent application CN201711358747.4). For information about the 11C12 heavy chain antibody, please refer to Chinese patent application CN201710120052.6. The sequence of the silica-binding peptide is shown in SEQ ID NO.9-13, and the present invention names it SBP1-5. In addition, the present invention also uses the silica-binding peptide CotB1P from the prior art (Abdelhamid, M.A., Motomura , K., Ikeda, T., Ishida, T., Hirota, R., & Kuroda, A. (2014). Affinity purification of recombinant proteins using a novel silica-binding peptide as a...

Embodiment 2

[0084] Example 2 Binding Kinetics Determination of Tailed Antibody Fragments Interacting with Quartz Surfaces

[0085] A new type of biosensor (FORTEBIO) was placed in a 70°C water bath oxidation solution (H 2 o 2 , a mixture of water and ammonia) for 30 minutes, and washed with ultrapure water to prepare a quartz surface. The prepared quartz sensor was used to measure the binding kinetics with a BLItz biolayer interferometer (FORTEBIO).

[0086] The specific determination steps of binding kinetics:

[0087] The binding kinetics of the silicon-based bound Nanobody constructs were determined using biolayer interferometry (BLItz (FORTEBIO)). Oxidize the liquid at 70°C (H 2 O2, water, ammonia (mixed 1:3:1) were incubated in a water bath for 30 minutes to prepare the silicon-based surface at the end of the quartz biosensor, and then washed with MilliQ water. Quartz biosensors were stored in 20 v / v % ethanol and pre-equilibrated in PBS for 10 minutes before use. Unless otherw...

Embodiment 3

[0093] Silica immobilization of the anti-CEA heavy chain antibody of embodiment 3

[0094] Silica nanoparticles and microspheres were ultrasonically dispersed in PBS for 5 minutes (Qsonica Q125). Add purified heavy chain antibodies to dispersed silica nanoparticles or microspheres. After a short vortex, incubate at room temperature for 30 minutes. The mixture was then centrifuged at 14800 rpm for 5 minutes to separate the particles. Wash twice with PBS and collect the supernatant from each washing step. The heavy chain antibodies immobilized in the particles or supernatant were quantified by SDS-PAGE and absorbance at 280nm.

[0095] Published silica-binding peptides (such as CotB1p, supra) have failed to identify polyhistidine as a key residue that enhances silica-binding affinity by forming hydrogen bonds with surface silanols. In the present invention, at least one histidine is added to promote hydrogen bond formation. The dissociation constant of one of the preferred po...

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Abstract

The invention discloses a polypeptide with silicon dioxide binding activity. The polypeptide contains a silicon dioxide binding unit forming a polypeptide main body part and a hydrogen bond forming unit formed by at least one histidine residue at the N-end or the C-end of the polypeptide. The invention also discloses a fusion protein containing the polypeptide and a functional protein with a biological effect, and a protein immobilization method. The fusion protein provided by the invention is non-covalently combined with directional control, so that related proteins are simply and quickly fixed on the surface of a silicon substrate in one step. Under the conditions of high salt, extreme pH and denaturation, protein binding is kept stable, peptide immobilization does not need surface modification or chemical coupling, and the fusion protein can be prepared at a low cost. Besides, the silicon dioxide binding peptide is fused to a specific tail end of the parent protein, so that the binding site of the protein is controlled, and the steric hindrance of the active site is eliminated.

Description

technical field [0001] The present invention relates to a protein immobilization method, specifically, the present invention relates to: (1) preparation of a fusion protein, which contains a polypeptide capable of binding to silica-based materials (quartz, glass). (2) A method for immobilizing the fusion protein. More specifically, the present invention relates to the above-mentioned proteins and methods, wherein the proteins are enzymes, antibodies, antibody fragments or antigens. Background technique [0002] Immobilization of functional proteins on solid substrates is a commercially important approach in the design and fabrication of bioanalytical tools (Vijayendran & Leckband, 2001; Weetall, 1993; Wu, Chen, & Liu, 2009). Functionalizing surfaces with high-value proteins such as enzymes, antibodies, antibody fragments, and antigens is a basic method for designing and manufacturing bioactive devices, which are increasingly used in biosensing, purification, detoxification,...

Claims

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Application Information

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IPC IPC(8): C07K17/14C07K16/30C12N11/14
CPCC07K17/14C07K16/3007C12N11/14C07K2317/569
Inventor 刘畅何利中宋海鹏
Owner 深圳市国创纳米抗体技术有限公司
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