Polypeptides and applications for protein surface immobilization

A fusion protein and sequence technology, applied in the protein field, can solve the problems of non-site-specific binding, increase the time and cost of the immobilization process, damage the protein, etc., and achieve the effect of enhancing binding affinity, eliminating steric hindrance and high binding strength.

Active Publication Date: 2022-06-21
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

AI Technical Summary

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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  • Polypeptides and applications for protein surface immobilization
  • Polypeptides and applications for protein surface immobilization
  • Polypeptides and applications for protein surface immobilization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 2

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

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

[0057] The silica-binding peptide was reconstituted into an anti-CEA heavy chain antibody (clone number 2D5, 11C12) isolated from immunized llamas following the procedure described in WO 94 / 25591. For information on 2D5 heavy chain antibody, see Chinese patent application CN201711358747.4). For information on the 11C12 heavy chain antibody, see Chinese patent application CN201710120052.6. The sequences of the silica-binding peptides are shown in SEQ ID NO. 9-13, which are named SBP1-5 in the present invention. 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 fusion tag. Applied mic...

Embodiment 2

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

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

[0086] Specific steps for the determination of binding kinetics:

[0087] The binding kinetics of the silicon-based bound Nanobody constructs were determined using biolayer interferometry (BLItz (FORTEBIO)). Oxidize liquid at 70°C (H 2 The silicon-based surface of the end of the quartz biosensor was incubated in a water bath for 30 minutes to prepare the silicon-based surface, 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 otherwise ind...

Embodiment 3

[0093] Example 3 Silica immobilization of tailed anti-CEA heavy chain antibodies

[0094] The silica nanoparticles and microspheres were ultrasonically dispersed in PBS for 5 minutes (Qsonica Q125). The purified heavy chain antibody is added to the 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 supernatant from each wash step. Heavy chain antibodies immobilized in particles or supernatants were quantified by SDS-PAGE and absorbance at 280 nm.

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

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Abstract

The present invention discloses a polypeptide with silica-binding activity. The polypeptide contains a silica-binding unit constituting the main part of the polypeptide and hydrogen composed of at least one histidine residue at the N-terminus or C-terminus of the polypeptide. bond forming unit. The invention also discloses a fusion protein containing the above-mentioned polypeptide and a functional protein with biological effects, and a protein immobilization method. The fusion protein provided by the invention enables related proteins to be fixed to the silicon-based surface simply, quickly and in one step through non-covalent combination with orientation control. Under high salt, extreme pH and denaturing conditions, protein binding remains stable, peptide immobilization requires neither surface modification nor chemical coupling, and fusion proteins can be prepared at a lower cost. Furthermore, since the silica-binding peptide is fused to one specific end of the parental protein, the binding site of the protein is controlled, eliminating steric hindrance to the active site.

Description

technical field [0001] The present invention relates to a protein immobilization method, in particular, the present invention relates to: (1) preparation of a fusion protein, the fusion protein containing a polypeptide capable of binding to a material (quartz, glass) based on silica. (2) A method of immobilizing the fusion protein. More specifically, the present invention relates to the aforementioned proteins and methods, wherein the proteins are enzymes, antibodies, antibody fragments or antigens. Background technique [0002] The immobilization of functional proteins on solid substrates is a method of commercial importance in the design and preparation of bioanalytical tools (Vijayendran & Leckband, 2001; Weetall, 1993; Wu, Chen, & Liu, 2009). Functionalizing surfaces with high-value proteins such as enzymes, antibodies, antibody fragments, antigens, etc. is a fundamental method for designing and fabricating bioactive devices, which are increasingly used in biosensing, p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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