CN110950963B

CN110950963B - Polypeptide for protein surface immobilization and application

Info

Publication number: CN110950963B
Application number: CN201911332672.1A
Authority: CN
Inventors: 刘畅; 何利中; 宋海鹏
Original assignee: Shenzhen Guochuang Nano Antibody Technology Co ltd
Current assignee: Shenzhen Guochuang Nano Antibody Technology Co ltd
Priority date: 2019-12-22
Filing date: 2019-12-22
Publication date: 2022-06-21
Anticipated expiration: 2039-12-22
Also published as: CN110950963A

Abstract

The invention discloses a polypeptide with silicon dioxide binding activity, which comprises a silicon dioxide binding unit forming the main body part of the polypeptide and a hydrogen bond forming unit consisting of at least one histidine residue at the N-terminal or C-terminal of the polypeptide. The invention also discloses a fusion protein containing the polypeptide and functional protein with biological effect, and a protein fixing method. The fusion protein provided by the invention is combined with the non-covalent of directional control, so that the related protein can be fixed on the silicon-based surface simply, quickly and in one step. 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 lower cost. In addition, since the silica binding peptide is fused to a specific end of the parent protein, the binding site of the protein is controlled, eliminating steric hindrance of the active site.

Description

Polypeptide for protein surface immobilization and application

Technical Field

The invention relates to a protein fixing method, in particular to: (1) preparation of fusion proteins containing polypeptides capable of binding to silica-based materials (quartz, glass). (2) A method of immobilizing the fusion protein. More specifically, the present invention relates to the above-mentioned protein and method, wherein the protein is an enzyme, an antibody fragment or an antigen.

Background

The immobilization of functional proteins on solid substrates is a commercially important method in the design and preparation of bioanalytical tools (Vijayendran & Leckband, 2001; Weetall, 1993; Wu, Chen, & Liu, 2009). Functionalization of surfaces with high value proteins such as enzymes, antibodies, antibody fragments, antigens, etc. is a fundamental method for designing and manufacturing biologically active devices, and is increasingly used in the industrial fields of biosensing, purification, detoxification, catalysis, etc.

Silica (silica) -based materials such as quartz and glass have various forms, smooth surfaces (coil & Baneyx,2014), nanoparticles (Ikeda et al, 2011), microspheres (Johnson, Zawadzka, deobalad, Crawford, & Paszczynski,2008), nanoparticles, and silicon-based surfaces are the most commonly used solid substrates for proteins due to their abundance, low cost, and biocompatibility. Although adsorption or chemical cross-linking can be used to immobilize high-value proteins. The improved method involves the use of recombinantly bound silica binding peptides. The fusion protein with the integrated silica binding peptide can be attached to a silicon-based surface through strong non-covalent interactions, and the immobilization method does not need to use a chemical cross-linking agent.

In general, the immobilization of proteins on silicon-based materials is based on the principles of nonspecific adsorption or chemical coupling. Adsorption to solid surfaces is usually accompanied by significant conformational disruption and partial unfolding and denaturation of the associated proteins (Hartmann, 2005; Lundqvist, Sethson, & Jonsson, 2004; Roach, Farrar, & Perry, 2006). For chemical coupling, protein immobilization is achieved by chemical modification of the surface (Chen, Kong, Yuan, & Fu, 2014; Wang, Rabe, Ahmed, & Niemeyer 2015), either by surface treatment with a functional silanol (e.g., 3-aminopropyltriethoxysilane) or by using an active chemical cross-linker (e.g., glutaraldehyde). Thus, this method has disadvantages in that (1) it requires the use of harmful chemicals, (2) it requires an additional reaction step, which increases the time and cost of the immobilization process, and (3) the uncontrolled orientation of immobilization may impair the function of the protein. For example, the binding site of an antibody may be blocked during chemical cross-linking because such binding is not site-specific.

The development of a rapid, low-cost and controllable target protein immobilization method is of great significance. The object of the present invention is to provide a polypeptide having silica-binding activity, so that a fusion protein containing these peptides can be immobilized on a silicon-based surface simply, rapidly, and in one step by non-covalent binding with orientation control.

Disclosure of Invention

In view of the above objects, the present invention provides, in a first aspect, a polypeptide having a silica-binding activity, which polypeptide comprises a silica-binding unit constituting a main portion of the polypeptide and a hydrogen bond-forming unit comprising at least one histidine residue at the N-terminus or C-terminus of the polypeptide.

The present invention is based on the discovery that the incorporation of a polyhistidine fragment into a fusion protein can promote the efficiency of silica binding to peptides, the polyhistidine fragment being capable of forming hydrogen bonds with silanol groups on the silica surface.

Based on the prior art theory (the binding strength of proteins to silica depends on charge, hydrogen bonding and polypeptide configuration), silica binding peptides were redesigned, introduced at the N-or C-terminus of the fusion protein to form a locally ordered structure independent of the folded protein. The presence of polyhistidine in the redesigned silica binding peptide can promote the formation of additional hydrogen bonds, thereby increasing the affinity of the fusion protein to the silica surface. The present invention also increases the possibility of protein immobilization to a surface by the design of structurally independent silica binding peptides, which are not necessary for the function of the protein, and thus can independently optimize the function of the binding protein and its surface binding properties. The silicon dioxide binding peptide comprises 3-30 amino acid residues, preferably 5-20 amino acid residues. The silica binding peptide comprises at least one, and preferably a plurality of positively charged amino acid residues capable of forming an electrostatic attraction with the silica surface. The silica binding peptide includes at least one, and preferably a plurality of histidine residues capable of forming hydrogen bonds with the silica surface.

In a preferred embodiment, the amino acid sequence of the hydrogen bond forming unit is selected from any one of the sequences shown in SEQ ID No. 1-3.

In a preferred embodiment, the amino acid sequence of the silica binding unit is selected from any one of the sequences shown in SEQ ID No. 4-8.

More preferably, the amino acid sequence of the polypeptide is selected from any one of the sequences shown in SEQ ID NO. 9-13.

Particularly preferred polypeptide fragments may be selected from the following amino acid sequences:

GRARAQRQSSRGRGGSHHHHHH (SEQ ID NO.9), defined herein as SBP 1;

GRARAQRQSSRAHHIHHIHH (SEQ ID NO.10), defined herein as SBP 2;

GRARAQRQSSRADAHHIHHIHH (SEQ ID NO.11), defined herein as SBP 3;

GRARAQRQSSRGRKSLSRADHIHHHHH (SEQ ID NO.12), defined herein as SBP 4;

DSARGFKKPGKRKSLSRADHIHHHHH (SEQ ID NO.13), defined herein as SBP 5.

It is understood that the inclusion of hhhhhhhhhh, hhihhh or portions of hhhhhhh amino acids in the sequence that are the core of the invention cannot be replaced or deleted by other conventional tags.

Secondly, the invention also provides a fusion protein containing the polypeptide, and the fusion protein also contains functional protein with biological effect.

The silica binding peptides of the invention may be introduced by mutation or insertion within the protein sequence or, preferably, may be added as a terminus at either or both of the N-or C-termini of the protein.

The sequence features promote the formation of an alpha-helical or beta-sheet conformation of the protein. The presence of the folded structure can be detected using conventional techniques such as nuclear magnetic resonance spectroscopy.

The present invention is applicable to proteins in general, and is not limited to any particular type. In a preferred embodiment, the functional protein comprises an enzyme, an antibody active fragment, an antigen.

According to an important embodiment of the present invention, antibodies or immunologically active fragments thereof can be immobilized on a solid surface to produce an immunologically active material with improved performance for use in immunological recognition procedures such as immunoaffinity techniques.

According to the common technical knowledge in the field, the antibody is an immunoglobulin which can be obtained naturally or synthesized artificially. Unless otherwise indicated, in the present specification, the terms "antibody" and "immunoglobulin" are used synonymously to each other. The immunologically active antibody fragments are part of a whole antibody that retains the ability to bind antigen activity. The antigen binding site may consist of the respective variable regions of the antibody light and heavy chains, or may consist of a single antibody variable region. Conventional fragments include Fab fragments, which comprise two binding sites linked together, Fv fragments (comprising the variable regions of the heavy and light chains of an antibody linked to one another), and single chain Fv fragments (the heavy and light chains of an antibody exist as fusion proteins).

According to a particular embodiment of the invention, said protein is an immunoglobulin naturally devoid of light chains (hereinafter referred to as heavy chain immunoglobulin), more particularly it is the variable region of an immunoglobulin naturally of heavy chains (VHH), also known as Nanobody (Nanobody), which is available from the family Camelidae (Hamers-Casterman et al, 1993), WO94/04678(Casterman et al). Alternatively, the protein may be a protein functionally equivalent to the above protein. An advantage of using immunoglobulins or immunoglobulin heavy chain variable regions derived from camelidae is that they are easy to produce on a large scale at low cost, e.g. using transformed lower prokaryotic hosts, such as e.coli.

In a preferred embodiment, the functional protein is a nanobody.

In a more preferred embodiment, the nanobody is a nanobody against a CEA antigen (carcinoembryonic antigen).

Particularly preferably, the nano antibody for the CEA antigen is 2D5 or 11C 12. The nano antibody 2D5 is disclosed in the Chinese patent application 201711358747.4, and the nano antibody 11C12 is disclosed in the Chinese patent application 201710120052.6.

Finally, the present invention provides a method for protein immobilization, which comprises contacting the above-mentioned fusion protein with a silica-based material, and allowing the fusion protein to be immobilized on the surface of the material.

It is to be understood that the present invention is applicable to proteins other than derived proteins such as antibodies. For example, enzymes may be coupled to a surface for juice clarification. Other applications of immobilized proteins can be found in Protein immobilization, pages 2-9, r.f. taylor ed., Marcel Dekker, inc., new york, 1991. The present invention may also be applied to diagnostic kits, as will be appreciated by those of ordinary skill in the art.

The advantages of the invention are embodied in the following aspects:

1. five novel silicon dioxide binding peptides can be fused with high-value proteins such as enzyme, antibody fragment, antigen and the like. In comparison to published silica-binding peptides (candle-Rochelle et al, 2012; Coyle & Baneyx, 2014; Ikeda et al, 2011), the present invention comprises at least one histidine to promote the formation of hydrogen bonds, thereby enhancing binding affinity (FIG. 1), and the fusion protein can target the surface of silica-based materials including quartz and glass by strong, rather than covalent bonds.

2. The silica binding peptide promotes high silica binding strength (kD ═ 3.6nM, table 1), the binding process does not require any chemical cross-linking agents or specific buffer environments

3. The direction of immobilization can be controlled by electrostatic attraction of the silica-binding peptide and formation of immobilized protein by hydrogen bonds, thereby minimizing functional interference with the modified protein. Since the silica binding peptide is fused to a specific end of the parent protein, the binding site of the protein is controlled and the steric hindrance of the active site is eliminated.

4. Immobilization of proteins by silica-binding peptides was stable in high salt, denaturing environments and a wide range of pH conditions (figure 3). Protein binding remains stable under high salt, extreme pH and denaturing conditions. The immobilization by means of the polypeptide requires neither surface modification nor chemical coupling, and the fusion protein can be prepared in one step by a relatively low-cost E.coli system.

5. Using recombinant technology, silica-binding peptides can be fused to functional proteins as a single molecule. The fusion protein can be economically expressed in an Escherichia coli system in only one step. This enables low cost functionalization and has potential applications in protein purification, protein immobilization, drug delivery, and bioanalysis.

6. The fusion protein of VHH and silica binding peptide was isolated from 5% BSA (bovine serum albumin) solution by silica microspheres using the single chain antibody fragment VHH model. This molecule was used to enrich and detect CEA on silica microspheres using a one-step capture scheme (fig. 4). The test device showed potential in achieving quantification of the CEA to ng range (fig. 5).

Drawings

FIG. 1 is a schematic representation of the synergistic effect of a polyhistidine peptide in enhancing silica binding affinity by promoting hydrogen bonding with surface silanes;

FIG. 2 is a schematic representation of the molecular design and sequence of anti-CEA nanobody binding silica binding peptide;

FIG. 3 is an SDS-PAGE profile of the clonal optimization of silica-binding peptide nanobody fusion protein expression;

FIG. 4 is an SDS-PAGE profile of silica-binding peptide nanobody fusion protein expression;

FIG. 5 shows SDS-PAGE patterns of fusion proteins obtained by one-step IMAC purification;

FIG. 6 is an SDS-PAGE pattern of the fusion protein of CotB1P with 2D5 and 11C12 after removal of the histidine tag;

FIG. 7 SDS-PAGE profile of enrichment of proteins displayed as MagSi-S particles using CotB 1P;

FIG. 8 SDS-PAGE profile of protein enrichment displayed as porous silica nanoparticles using CotB 1P;

FIG. 9 is an SDS-PAGE pattern of binding assays of SBP4 and SBP5 to silica particles;

FIG. 10 is an SDS-PAGE pattern of binding assay between SBP4 and SBP5 and silica particles;

FIG. 11 is a typical Nanobody HPLC chromatogram performed using a C5 column;

FIG. 12 is a schematic of the silicon binding affinity of fusion proteins at different pH and salt concentrations;

FIG. 13 is a schematic diagram of the use of the fusion protein in CEA detection;

FIG. 14.520nm fluorescence intensity vs. CEA solution concentration diagram;

FIG. 15 is a fluorescence microscopic image of the silica microspheres isolated in the assay;

FIG. 16 is a control graph of assays for determining complementary binding antibody affinity activity using the 2D5-SBP4 fusion protein.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection of the invention as defined by the claims.

Example 1 preparation of silica binding peptide anti-CEA Nanobody fusion proteins

1. Construction of silica-binding peptide anti-CEA nanobody fusion protein

The silica binding peptide was recombined 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 on the 2D5 heavy chain antibody see chinese patent application CN 201711358747.4). The information on the 11C12 heavy chain antibody is described in chinese patent application CN 201710120052.6. The sequence of the silica binding peptide is shown in SEQ ID NO.9-13, which the present invention designates as SBP1-5, and in addition, the present invention uses a fusion protein of the silica binding peptide CotB1P (Abdelhamid, M.A., Motomomura, K., Ikeda, T., Ishida, T., Hirota, R., & Kuroda, A. (2014.) Affinity purification of recombinant proteins using a non-binding peptide a fusion tag.applied microbiology and biotechnology,98(12),5677-5684.) from the prior art with 2D5,11C12 nm antibody for comparative studies. Both "heavy chain antibody" and "nanobody" are used herein to refer to an antibody that naturally lacks a light chain.

The SWISS-MODEL is applied to predict the structure of the anti-CEA nano antibody according to a highly conserved framework region in an amino acid sequence. The PEP-FOLD3.5 server was used again to predict the silicon-binding peptide structure. Proteins and peptides were visualized using PyMOL.

The DNA containing the final construct with pet28a (+) plasmid as backbone plasmid was synthesized by GenScript, and 4. mu.g of the constructed plasmid was resuspended in 30. mu.L dH₂O, and transformed into Escherichia coli.

FIG. 1 is a schematic representation of the synergistic effect of a polyhistidine peptide in enhancing silica binding affinity by promoting hydrogen bonding with surface silanes; figure 1 illustrates that histidine plays a key role in facilitating immobilization of peptides. In the figure 1-a, 1 is His-6 label, 2 is thrombin cleavage site, 3 is nano antibody, 4 is connecting peptide, and 5 is CotB1 p. CotB1P is a 14 amino acid long, arginine-rich polypeptide sequence with strong affinity for silicon-based surfaces. This sequence was found by Abdelhamid, m.a., motomra, k., Ikeda, t., Ishida, t., Hirota, r. & Kuroda, a. in 2014.

In FIG. 1-B, A is a fusion protein of a nano antibody-CotB 1p with a histidine tag, and B is a fusion protein of a nano antibody-CotB 1P with a histidine tag cut off. FIG. 1-b illustrates that the nanobody with the sequence of CotB1P is significantly enriched on the quartz surface.

In FIG. 1-c, A is a fusion protein of a nano antibody-CotB 1P with a histidine tag, and B is a fusion protein of a nano antibody-CotB 1P with a histidine tag cut off. FIG. 1-c illustrates that the nanobody with the sequence of CotB1P and the N-terminal histidine tag is significantly enriched on the surface of silicon nanoparticles, and the binding force of the nanobody on the silicon surface is reduced after the histidine tag is removed.

In FIG. 1-d, A is a fusion protein of a nano antibody-CotB 1P with a histidine tag, B is a fusion protein of a nano antibody-CotB 1P with a cut-out histidine tag, and FIG. 1-d illustrates that the binding force of the nano antibody with CotB1p to silicon is remarkably reduced after an N-terminal histidine sequence is cut by enzyme.

In FIG. 1-e, A is 500mM NaCl, B is 250mM NaCl,50mM CuCl₂. Fig. 1-e illustrates that the nanobody of Cu2+ ion inhibited histidine at consistent ionic strength showed a significant decrease in silicon surface binding force.

FIG. 2 shows a schematic diagram of the structure of silica-binding peptide anti-CEA nanobody fusion protein, and the sequence information and spatial configuration prediction of the silica-binding peptide used in the present invention. Wherein, FIG. 2(a) is a schematic molecular design diagram of anti-CEA nanobody binding to silica binding peptide; FIG. 2(b) is a schematic representation of the amino acid residue arrangement of the silica-binding peptide; FIG. 2(c) is a schematic diagram of the design structure of the silica-binding peptide.

The information reference of Car9 in FIG. 2(c) (style, B.L., & Baneyx, F. (2014.). A clean silicon-binding affinity tag for Rapid and antibiotic protein purification, 111(10),2019-2026.)

2. Expression and purification of fusion proteins

The anti-CEA heavy chain antibody containing the silica binding peptide was expressed in E.coli SHuffle T7.

Clones from freshly transformed E.coli were plated on LB and TB plates, the plates were incubated at 30 ℃ for 36-48 hours, individual colonies of each heavy chain antibody type were "scraped" from the plates and inoculated into 10ml TB medium seed cultures, and 10ml cultures were incubated at 30 ℃ for 12 hours. The seed cultures were re-cultured in large shake flasks for protein expression according to the NEB Shuffle strain expression instructions.

The heavy chain antibody seed culture was inoculated into 500mL TB medium in 2L shake flasks with an initial OD of 0.1. Protein expression was induced by 1mM IPTG, after which the flasks were incubated at 20 ℃ for 36-48 hours. Coli cells were collected by centrifugation at 10000rpm and lysed by sonication in lysis buffer (20mM Tris-HCl, pH8,500mM NaCl,1mM EDTA, 0.5% Triton-X). The cell lysate samples were centrifuged again at 10000rpm for 30 minutes and the supernatant was filtered through a 0.22 μm filter before purification.

Nanobodies were purified using HisTrap Ni-NTA column (GE healthcare) which binds 4mg his-tag protein per ml capacity. Typically, a 1mL HisTrap HP column is first equilibrated in 20mM Tris-HCl pH8,500mM NaCl for 5 column volumes, and then the filtered supernatant is added. After loading the sample on the HisTrap column, the column was washed with 20mM Tris-HCl pH8,500mM NaCl for 7 column volumes and eluted with elution buffer (20mM Tris-HCl pH8,500mM NaCl,500mM imidazole).

Thrombin was used to cleave the histidine tag at the N-terminus of the nanobody at the thrombin cleavage site. Briefly, 2. mu.L of thrombin at a concentration of 1U/. mu.L was added to a protein solution at a concentration of 0.25mg/mL and incubated overnight at 4 ℃. The cleaved nanobodies were purified by desalting in PBS (phosphate buffered saline) using a 5mL desalting column (GE healthcare). The cleavage of the nanobody was confirmed by SDS-PAGE and MALDI-TOF mass spectrometry.

Heavy chain antibodies were eluted by washing over 12 column volumes with a linear gradient of 0-0.5M imidazole, followed by desalting into PBS through a 53mL Sephadex G25 desalting column. The purity of the heavy chain antibody isolated by this method can reach 95% or more. Fig. 11 is a typical nanobody HPLC chromatogram performed using a C5 column. Mobile phase: buffer a, water 0.1% TFA; eluent B, 100% cyanomethane, 0.1% TFA. In HPLC peak shape, it can be concluded that the nanobody purified by one-step nickel column has a purity of 95% or more and elutes at 19 minutes.

SDS-PAGE was performed according to the conventional procedure. A preformed 4-12% Bris-Tris gel (ThermoFisher Scientific) was used. Approximately 50ug of silicon nanoparticles were dispersed in 20 μ L of standard reducing sample buffer, incubated at 95 ℃ for 10 minutes and loaded directly into the gel wells.

FIG. 3 is an SDS-PAGE profile of the clonal optimization of the expression of silica-binding peptide nanobody fusion proteins. The sample addition was 10. mu.l, wherein lane 1:2D5-SBP2 culture precipitate (clone A), lane 2:2D5-SBP2 culture supernatant (clone A), lane 3:2D5-SBP2 culture precipitate (clone B), lane 4:2D5-SBP2 culture supernatant (clone B), lane 5:2D5-SBP2 culture precipitate (clone A), lane 6:2D5-SBP2 culture supernatant (clone A), lane 7: Nb-L2 culture precipitate (clone A), lane 8: Nb-L2 culture supernatant (clone A), lane 9: Nb-L2 culture precipitate (clone B), lane 10: Nb-L9 culture supernatant (clone B), lane 11:11C 695695695695695 12-SBP2 culture precipitate (clone 84A), lane 12:11C 8653-SBP 2 culture Supernatant (SBA), lane 13: SBC 86 2-SBP 86 2 culture precipitate (clone B), lane 14:11C12-SBP2 culture supernatant (clone B). L2 of Nb-L2 in lanes 7-9 refers to ribosomal protein L2, L2 is another protein motif with silicon-based binding ability, which is not preferred due to low expression levels and does not meet low cost production requirements.

FIG. 4 is an SDS-PAGE pattern of silica-bound peptide nanobody fusion protein expression. The sample addition was 10. mu.l, wherein lane 1:2D5-SBP1 culture supernatant, lane 2:2D5-SBP1 culture supernatant, lane 3:2D5-SBP4 culture precipitate, lane 4:2D5-SBP4 culture supernatant, lane 5:2D5-SBP5 culture precipitate, lane 6:2D5-SBP5 culture supernatant.

FIGS. 3 and 4 illustrate that the designed polypeptide can be expressed in E.coli.

FIG. 5 is an SDS-PAGE profile of the fusion protein obtained by one-step purification of IMAC. FIG. 5 provides protein purity from a single purification step of Ni-IMAC, in lane 1:2D5-SBP2, lane 2:2D5-SBP3, lane 3:2D5-SBP4, lane 4:2D5-SBP 5.

FIG. 6 is an SDS-PAGE pattern of the fusion protein of CotB1P and 2D5 and 11C12 after removal of the histidine tag. Of these, lane 1:11C12-CotB1P was cleaved by thrombin at pH6.2, lane 2:2D5-CotB1P with the histidine tag removed (the N-terminal histidine tag was cleaved by thrombin) lane 3:2D 5-CotB1P histidine tag was cleaved by thrombin at pH6.2, and lane 4:2D5-CotB1P histidine tag was cleaved by thrombin at pH 7.4. FIG. 6 is supporting data illustrating that in FIG. 1-d histidine is completely removed by thrombin.

FIG. 7 illustrates the enrichment of proteins displayed as MagSi-S particles using the silica binding peptide CotB 1P. The binding force is reduced after removing the poly-histidine. In fig. 7, lane 1:11C12-CotB 1P0.13mg/ml, lane 2:11C12-CotB1P washed once with high salt PBS, lane 3:11C12-CotB1P washed twice with high salt PBS, lane 4:11C12-CotB1P on pure magnesium, lane 5: 11C12-CotB1P 0.13.13 mg/ml, the histidine tag was excised, lane 6:11C12-CotB1P high salt PBS was washed once, the histidine tag was excised, lane 7:11C12-CotB1P high salt PBS was washed twice, the histidine tag was excised, lane 8:11C12-CotB1P was over pure magnesium, the histidine tag was excised, lane 9:2D 5-CotB1P 0.13.13 mg/ml, lane 10:2D5-CotB1P washed once with high-salt PBS, lane 11:2D5-CotB1P washed twice with high-salt PBS, lane 12:2D5-CotB1P on pure magnesium ions, lane 13:2D 5-CotB1P 0.13.13 mg/ml, the histidine tag was excised, lane 14:2D5-CotB1P was washed once with high salt PBS and the histidine tag was excised, lane 15:2D5-CotB1P was washed twice with high salt PBS and the histidine tag was excised, lane 16:2D5-CotB1P was on top of pure magnesium and the histidine tag was excised. FIG. 7 additionally demonstrates that for the preferred nanobodies 2D5 and 11C12 in FIGS. 1-C and 1-D, removal of the N-terminal histidine sequence resulted in a decrease in binding force to silicon nanoparticles

FIG. 8 illustrates the enrichment of proteins displayed as porous silica nanoparticles (particle size 200nm, pore size 4nm) using the silica-binding peptide CotB 1P. In fig. 8, lane 1:

11C12-CotB1P 0.13.13 mg/ml, lane 2:11C12-CotB1P on silica ion, lane 3:11C12-CotB1P washed once with high salt PBS, lane 4:11C12-CotB1P washed twice with high salt PBS, lane 5: 11C12-CotB1P 0.13.13 mg/ml, the histidine tag was excised, lane 6:11C12-CotB1P high salt PBS was washed once, the histidine tag was excised, lane 7:11C12-CotB1P high salt PBS was washed twice, the histidine tag was excised, lane 8:11C12-CotB1P was over silica ion, the histidine tag was excised, lane 9:

2D5-CotB1P 0.13.13 mg/ml, lane 10:2D5-CotB1P high salt PBS wash once, lane 11:2D5-CotB1P high salt PBS wash twice, lane 12:2D5-CotB1P on silica ion, lane 13:2D 5-CotB1P 0.13.13 mg/ml, the histidine tag was excised, lane 14:2D5-CotB1P was washed once with high salt PBS and the histidine tag was excised, lane 15:2D5-CotB1P was washed twice with high salt PBS and the histidine tag was excised, lane 16:2D5-CotB1P was on silica and the histidine tag was excised.

Example 2 binding kinetics assay of interaction of tailed antibody fragments with Quartz surfaces

Placing a new biosensor (FORTEBIO) in 70 deg.C water bath oxidation solution (H)₂O₂A 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 binding kinetics with a BLItz bio-layer interferometer (FORTEBIO).

Specific assay procedure for binding kinetics:

the binding kinetics of silica-based binding nanobody constructs were determined using biolayer interferometry (blitz (fortebio)). Oxidation of the liquid (H) at 70 deg.C₂O2, water, ammonia mixed at 1:3: 1) water bath were incubated on the silica biosensor terminal silicon-based surface for 30 minutes to prepare a silicon-based surface, followed by washing with MilliQ water.Quartz biosensors were stored in 20 v/v% ethanol and pre-equilibrated with PBS for 10 min prior to use. Unless otherwise indicated, all kinetic determinations were performed in PBS and kinetic calculations were performed using BLItz Pro 1.2 software (FORTEBIO).

Kinetic measurements were performed in PBS or 20mm phosphate buffer at different concentrations of NaCl. Kinetic data analysis used BLItz Pro 1.2 software (FORTEBIO). Table 1 shows the results of measuring the quartz binding affinity of the silica binding peptides fused to VHH using a biolayer interferometer.

TABLE 1 detection of binding kinetics of silica affinity peptides

The table illustrates the binding kinetic constants determined by BLItz, demonstrating that the designed polypeptide SBP1-5 has a significant silica binding capacity increase compared to CotB1 p. With the strongest SBP4 being improved by about 100 times.

FIG. 12 is a graph showing the silicon binding affinity of the fusion protein at various pH and salt concentrations; in FIG. 12, Kon is an Association constant (Association constant) and Koff is a dissociation constant (dissociation constant).

Example 3 silica immobilization of tailed anti-CEA heavy chain antibody

The silica nanoparticles and microspheres were placed in PBS and ultrasonically dispersed for 5 minutes (Qsonica Q125). The purified heavy chain antibody is added to dispersed silica nanoparticles or microspheres. After short vortexes, incubate for 30 min at room temperature. The mixture was then centrifuged at 14800rpm for 5 minutes to separate the particles. Washed twice with PBS and the supernatant from each washing step was collected. The heavy chain antibody immobilized in the particles or supernatant was quantitatively analyzed by SDS-PAGE and absorbance at 280 nm.

The currently published silica binding peptides (e.g., CotB1p, references supra) fail to identify polyhistidine as a key residue to enhance 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 polypeptide silica binding peptides (SBP4) was increased by about 100-fold as compared to control CotB1p as determined by optical interferometry.

FIG. 9 illustrates the use of SBP4 and SBP5 to improve binding to silica particles. In fig. 9, lane 1:2D5-SBP 4 solution (0.34mg/ml), lane 2:2D5-SBP4 supernatant, lane 3:2D5-SBP4 PBS wash once, lane 4:2D5-SBP4 PBS wash twice, lane 5: uptake of 2D5-SBP4 on 0.5mg particles, lane 6:2D5-SBP5 solution (0.29mg/ml), lane 7:2D5-SBP5 supernatant, lane 8:2D5-SBP5 PBS wash once, lane 9:2D5-SBP5 PBS wash twice, lane 10: absorption of 2D5-SBP5 on 0.5mg particles. FIG. 9 illustrates that the preferred SBP4 and SBP5 allow for substantial enrichment of nanobodies on the surface of silicon nanoparticles

FIG. 10 illustrates that silica binding peptides facilitate the immobilization of proteins on silica particles.

In fig. 10, lane 1:11C12-SBP 4, lane 2:11C12-SBP4 supernatant, lane 3:11C12-SBP4 PBS wash once, lane 4:11C12-SBP4 PBS wash twice, lane 5: 11C12-SBP4 on particles (0.1mg), lane 6:2D5-SBP 4, lane 7:2D5-SBP5 supernatant, lane 8:2D5-SBP5 PBS wash once, lane 9:2D5-SBP5 PBS wash twice, lane 10:2D 5-SBP5 on particles (0.1mg), lane 11:2D 5-SBP4, lane 12:2D5-SBP4 supernatant, lane 13:2D5-SBP4 PBS wash once, lane 14:2D5-SBP4 PBS wash twice, lane 15:2D 5-SBP4 on particles (0.1 mg).

Mutations to the silica-binding peptide, including substitutions, additions or deletions of one or more amino acids, may result in a significant change in the silica-binding capacity. The key factors in the design of silica-binding peptides are 1) the positively charged residues (arginine, lysine) used to establish electrostatic interactions. 2) Polyhistidine allows hydrogen bonding to form. 3) Residues with targeting function ensure maximum performance.

Example 4 application of fusion protein in CEA detection

The fusion protein of VHH and silica binding peptide was isolated from 5% BSA solution using a single chain antibody fragment VHH model by silica microspheres. This molecule was used to enrich and detect CEA on silica microspheres using a one-step capture scheme. The detection device shows potential in realizing quantitative determination of the CEA to ng range.

The method comprises the following specific steps:

mix 20. mu.L of 2D5-SBP4 at a concentration of 100. mu.g/mL, 20. mu.L of 10. mu.g/mL ATTO488 fuel-labeled secondary nanobody, and CEA antigen at different concentrations (10-10000ng/mL) in a centrifuge tube. The mixed sample was incubated at room temperature for 10 minutes, then 25. mu.g of silicon nanoparticles were added to mix and incubation continued for 10 minutes. After centrifugation, the silicon nanoparticles were washed 2 times with PBS buffer to remove the unfixed antigen-antibody complex. The washed silicon nanoparticles were redispersed in 200 μ L PBS and the fluorescence signal was measured at 535nm using a microplate reader (Tecan Infinite 200 pro).

Second Nanobody fluorescent Label

A2 mg/mL NHS-ATTO488 stain was prepared in DMSO. The second nanobody was concentrated to 1mg/mL using ultrafiltration prior to labeling. 50 μ L of concentrated Nanobody requires the addition of 3 μ L of NHS-ATTO488 and 7 μ L of 1M NaHCO₃. The labeling reaction was carried out overnight at 4 ℃ and the product was purified by standard methods using a MiniTrap desalting column (GE healthcare).

FIG. 13 is a schematic diagram of the application of the fusion protein to the detection of CEA;

FIG. 14.520nm fluorescence intensity vs. CEA solution concentration.

FIG. 15 is a fluorescence microscopy micrograph of the isolated silica microspheres of FIG. 14, wherein BF is bright field and the fluorescence image is taken at 250ms exposure.

FIG. 16 is a test pattern for determination of complementary binding antibodies using 2D5-SBP4 fusion protein; wherein, 2D5-SBP4 is fixed on the surface of the biological layer interference sensor. The affinity activity of the second nanobody can be determined by capturing 600nM CEA in solution.

Sequence listing

<110> Shenzhen Shang Nanobody technology Limited

<120> polypeptide for protein surface immobilization and application

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

His His His His His His

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

His His Ile His His Ile His His

1 5

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

His Ile His His His His His

1 5

<210> 4

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Gly Arg Gly Gly Ser

1 5 10 15

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Ala

1 5 10

<210> 6

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Ala Asp Ala

1 5 10

<210> 7

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Gly Arg Lys Ser Leu

1 5 10 15

Ser Arg Ala Asp

20

<210> 8

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ser Ala Arg Gly Phe Lys Lys Pro Gly Lys Arg Lys Ser Leu Ser

1 5 10 15

Arg Ala Asp

<210> 9

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Gly Arg Gly Gly Ser

1 5 10 15

His His His His His His

20

<210> 10

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Ala His His Ile His

1 5 10 15

His Ile His His

20

<210> 11

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Ala Asp Ala His His

1 5 10 15

Ile His His Ile His His

20

<210> 12

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Arg Ala Arg Ala Gln Arg Gln Ser Ser Arg Gly Arg Lys Ser Leu

1 5 10 15

Ser Arg Ala Asp His Ile His His His His His

20 25

<210> 13

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Ser Ala Arg Gly Phe Lys Lys Pro Gly Lys Arg Lys Ser Leu Ser

1 5 10 15

Arg Ala Asp His Ile His His His His His

20 25

Claims

1. A polypeptide having a silica-binding activity, which comprises a silica-binding unit constituting a main portion of the polypeptide and a hydrogen bond-forming unit consisting of at least one histidine residue at the N-terminus or C-terminus of the polypeptide, wherein the amino acid sequence of the hydrogen bond-forming unit is selected from any one of the sequences represented by SEQ ID nos. 1 to 3, and the amino acid sequence of the silica-binding unit is selected from any one of the sequences represented by SEQ ID nos. 4 to 8.

2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is selected from any one of the sequences shown in SEQ ID nos. 9-13.

3. A fusion protein comprising the polypeptide of claim 1 or 2, wherein said fusion protein further comprises a functional protein having a biological effect.

4. The fusion protein of claim 3, wherein the functional protein comprises an immunologically active fragment, a receptor or binding fragment thereof, a lectin, and an enzyme.

5. The fusion protein of claim 4, wherein the functional protein is a nanobody.

6. The fusion protein of claim 5, wherein the nanobody is a nanobody against a CEA antigen.

7. The fusion protein of claim 6, wherein the nanobody against CEA antigen is 2D5 or 11C12, wherein the information of nanobody 2D5 is disclosed in Chinese patent application CN201711358747.4, and the information of nanobody 11C12 is disclosed in Chinese patent application CN 201710120052.6.

8. A method for immobilizing a protein, comprising contacting the fusion protein according to any one of claims 4 to 7 with a silica-based material and immobilizing the fusion protein on the surface of the material.