AU2020103565A4 - Anti-His single domain antibodies and use thereof - Google Patents

Anti-His single domain antibodies and use thereof Download PDF

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AU2020103565A4
AU2020103565A4 AU2020103565A AU2020103565A AU2020103565A4 AU 2020103565 A4 AU2020103565 A4 AU 2020103565A4 AU 2020103565 A AU2020103565 A AU 2020103565A AU 2020103565 A AU2020103565 A AU 2020103565A AU 2020103565 A4 AU2020103565 A4 AU 2020103565A4
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Xiaokang Bai
Jinde Chen
Xuerou Wang
Ziyang Wang
Liqun Xiao
Qizhuang Ye
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Next Medicine Co Ltd
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Abstract

We disclose six single domain antibodies that specifically bind to proteins with His tag. The anti-His single domain antibodies were expressed in E. coli and purified to near 5 homogeneity, demonstrating their easy production as binding reagents. These proteins, with a range of binding ability and specificity, will find their use in identification, quantitation, and purification of His-tagged proteins by themselves or in covalently or non-covalently modified forms. Figures FR1 CDR1 FR2 QVQLQESGGGLVQAGGSLRLSCAASGS ISNFDSMG WYRQAPG H313 QVQLQESGGGLVQAGGSLRLSCAASGS ISYTRNMG WYRQAPG H1216 QVQLQESGGGLVQAGGSLRLSCAASGY IFLRHYMG WYRQAPG H4333 QVQLQESGGGLVQAGGSLRLSCAASGS ISLEAEMG WYRQAPG H4352 QVQLQESGGGLVQAGGSLRLSCAASGY IFTSASMG WYRQAPG H4447 QVQLQESGGGLVQAGGSLRLSCAASGS ISLEAEMG WYRQAPG H7219 FR2 CDR2 FR3 KERELVAT INSGATTNYADSVKG RFTISRDNAKNTVYLQMNS H313 KERELVAA INYGTTTYYADSVKG RFTISRDNAKNTVYLQMNS H1216 KERELVAA ISSGTNTYYADSVKG RFTISRDNAKNTVYLQMNS H4333 KERELVAT ISPGSITYYADSVKG RFTISRDNAKNTVYLQMNS H4352 KERELVAT INAGTNTYYADSVKG RFTISRDNAKNTVYLQMNS H4447 KERELVAG ISRGTTTYYADSVKG RFTISRDNAKNTVYLQMNS H7219 FR3 CDR3 FR4 LKPEDTAVYYC AASHYYHLILSFQLSY WGQGTQVTVSS H313 LKPEDTAVYYC AVLSSKSHKYIYRFYY WGQGTQVTVSS H1216 LKPEDTAVYYC AVELFPRPYRYLGMINLVY WGQGTQVTVSS H4333 LKPEDTAVYYC AVLGPALMMAIDDRAMDFIY WGQGTQVTVSS H4352 LKPEDTAVYYC AVGWVWGAPWLHTIAAVFGY WGQGTQVTVSS H4447 LKPEDTAVYYC AVPHAIHSPARIIYFLHLMY WGQGTQVTVSS H7219 Figure 1

Description

Figures
FR1 CDR1 FR2 QVQLQESGGGLVQAGGSLRLSCAASGS ISNFDSMG WYRQAPG H313 QVQLQESGGGLVQAGGSLRLSCAASGS ISYTRNMG WYRQAPG H1216 QVQLQESGGGLVQAGGSLRLSCAASGY IFLRHYMG WYRQAPG H4333 QVQLQESGGGLVQAGGSLRLSCAASGS ISLEAEMG WYRQAPG H4352 QVQLQESGGGLVQAGGSLRLSCAASGY IFTSASMG WYRQAPG H4447 QVQLQESGGGLVQAGGSLRLSCAASGS ISLEAEMG WYRQAPG H7219
FR2 CDR2 FR3 KERELVAT INSGATTNYADSVKG RFTISRDNAKNTVYLQMNS H313 KERELVAA INYGTTTYYADSVKG RFTISRDNAKNTVYLQMNS H1216 KERELVAA ISSGTNTYYADSVKG RFTISRDNAKNTVYLQMNS H4333 KERELVAT ISPGSITYYADSVKG RFTISRDNAKNTVYLQMNS H4352 KERELVAT INAGTNTYYADSVKG RFTISRDNAKNTVYLQMNS H4447 KERELVAG ISRGTTTYYADSVKG RFTISRDNAKNTVYLQMNS H7219
FR3 CDR3 FR4 LKPEDTAVYYC AASHYYHLILSFQLSY WGQGTQVTVSS H313 LKPEDTAVYYC AVLSSKSHKYIYRFYY WGQGTQVTVSS H1216 LKPEDTAVYYC AVELFPRPYRYLGMINLVY WGQGTQVTVSS H4333 LKPEDTAVYYC AVLGPALMMAIDDRAMDFIY WGQGTQVTVSS H4352 LKPEDTAVYYC AVGWVWGAPWLHTIAAVFGY WGQGTQVTVSS H4447 LKPEDTAVYYC AVPHAIHSPARIIYFLHLMY WGQGTQVTVSS H7219
Figure 1
Anti-His single domain antibodies and use thereof
Technical field The field of this invention is biological technology. More specifically, antibodies and binding reagents used in protein identification, quantitation, and purification.
Background Proteins have many functions, including structure, signal molecules, carriers, and enzymes, and they play vital roles in biological processes. Protein identification, quantitation, and purification are essential in elucidation of their biological functions. To facilitate the process, proteins are often tagged with a short peptide sequence. These protein tags include polyHis tag (poly-histidine tag, 6X His; or simply called His tag), HA tag, Myc tag, FLAG tag, etc. (CL Young, ZT Britton, AS Robinson, Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnol J 2012, 7, 620 634). His tag is the most widely used affinity tag for purifying recombinant proteins. It has small size and can position at either the N- or C-terminus, and purification can be carried out under both native and denaturing conditions. The His-tagged proteins can be purified on immobilized metal-affinity chromatography (IMAC), based on the interaction between two histidine 2 residues in the His tag and the transition metal ion (Ni , Co 2 , Cu2 , Zn2 ) immobilized on a matrix. Antibody is the protein with superb specificity to interact with its antigen, and anti-His antibodies have been identified and commercially available. Antibodies, including anti-His antibodies, mentioned here are conventional antibodies, which are either monoclonal or polyclonal generated by immunizing animals. Anti-His antibodies have been used widely in protein identification ranging from Western blotting and ELISA assays to microscopic imaging. However, anti-His antibodies have not been used in purification of proteins with His tag, and the reasons include their high cost of production and their lowthermal and chemical stabilities. Single domain antibodies (or called nanobodies, VHH) disclosed here are different from conventional antibodies. Single domain antibodies are the binding molecules about 10-fold smaller than the conventional antibodies (15 kDa vs. 150 kDa), while maintaining the antigen binding affinity and specificity (D Saerens, GH Ghassabeh, S Muydermans, Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 2008, 8, 600-608; E Beghein, J Gettemans, Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein-Protein Interaction Analysis, and Protein Function Exploration. Front Immunol 2017, 8, 771). The advantages of single domain antibodies, in comparison with conventional antibodies, including low-cost production in E. coli, high thermal stabilities, and easy chemical modifications. Single domain antibodies can potentially replace conventional antibody not only in protein identification and quantitation, but also in protein purification. We have carried out screening of a synthetic phage library for single domain antibodies that specifically bind to recombinant proteins with His tag. Now we disclose six such antibodies with high affinity and specificity for proteins with His-tag and their use in protein identification, quantitation, and purification.
Summary The object of the present disclosure is to provide six single domain antibodies that specifically bind recombinant proteins with His-tag. Their native or modified forms, or their fusion proteins, can be used in identification, quantitation, and purification of proteins with His tag. In the first aspect of the present disclosure, it provides the six single domain antibodies, and each one of them has three complementary determining regions CDR1, CDR2, and CDR3, separated by framework regions FRI, FR2, FR3, and FR4, as in a typical single domain antibody. Each of Said single domain antibodies contains the CDR1, CDR2, CDR3 combination of the sequences defined by SEQ ID NOS: 1, 2, and 3; SEQ ID NOS: 4, 5, and 6; SEQ ID NOS: 7, 8, and 9; SEQ ID NOS: 10, 11, and 12; SEQ ID NOS: 13, 14, and 15; or SEQ ID NOS: 16, 17, and 18. In one preferred embodiment, the CDR1, CDR2, CDR3 combination has sequences with identity of 80% or more to one of said CDR1, CDR2, CDR3 combinations of sequences. In the second aspect of the present disclosure, it provides the six single domain antibodies, and each of said single domain antibodies contains the polypeptide sequence defined by SEQ ID NOS: 19, 20, 21, 22, 23, or 24. In one preferred embodiment, the polypeptide sequence has a sequence with identity of 80% or more to said polypeptide sequences. In the third aspect of the present disclosure, it provides a fusion protein between the single domain antibody according to the first, or the second aspect of the present disclosure, and an additional sequence. In one preferred embodiment, said additional sequence is selected from the group of short peptides, comprising signal peptide, affinity tag, and amino acid residues with chemically modifiable amino, carboxyl, or thiol groups. In another preferred embodiment, said additional sequence is selected from the group of binding proteins, comprising another single domain antibody, scFv, Fab, avidin, and streptavidin. In another preferred embodiment, said additional sequence is selected from the group of fluorescent proteins, such as green fluorescent protein. In another embodiment, said additional sequence is selected from the group of enzymes commonly used in detection and quantitation, comprising luciferase, alkaline phosphatase, horseradish peroxidase, and sortase. The fourth aspect of the present disclosure provides the antibodies according to the first, the second, or the third aspect of the present disclosure, that are modified covalently or non covalently by a label. In one preferred embodiment, said label comprises chemical dyes with various colors, such as malachite green, and Congo red. In another preferred embodiment, said label comprises fluorescent compounds, such as fluorescein and rhodamine. In another preferred embodiment, said label comprises luminogenic compounds, such as luminol, and acridinium esters. In another preferred embodiment, said label comprises gold particles, such as colloidal gold used in lateral flow immunochromatography. In another preferred embodiment, said label comprises biotin, and biotin analogs and derivatives. In another embodiment, said label is selected from the group of enzymes, comprising luciferase, alkaline phosphatase, and horseradish peroxidase. The fifth aspect of the present disclosure provides attachment of the antibodies according to the first, the second, or the third aspect of the present disclosure to a solid support. In one preferred embodiment, said support comprises polymer membranes used in separation, filtration, and blotting, such as polyvinylidene fluoride (PVDF), nitrocellulose (NC), and nylon. In another preferred embodiment, said support comprises various resins and packing materials for chromatography. In another preferred embodiment, said support comprises various bead-formed materials, such as polystyrene beads, and magnetic beads. The sixth aspect of the present disclosure provides use of the antibodies according to the third, the fourth, or the fifth aspect of the present disclosure. In one preferred embodiment, said use comprises identification and quantitation of proteins such as in Western blotting and ELISA assays, and purification of proteins such as using chromatographic columns and magnetic beads. These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and claims.
Description of the drawings
Figure 1. This drawing is to disclose protein sequences of the six single domain antibodies with affinity towards the His tag of recombinant proteins. Three complementary determining regions CDR1, CDR2, and CDR3 are indicated and emphasized in bald faces. Four framework regions FRI, FR2, FR3, and FR4 are also indicated. Figure 2. This drawing is to show the protein sizes and purities of the six single domain antibodies, characterized by polyacrylamide gel electrophoresis (PAGE). The sizes of protein markers are indicated. This drawing is also to show the size and purity, after expression and purification, of the single domain antibody His1, which has thiol groups added to the C terminus for easy chemical modifications. Figure 3. This drawing is to show the ELISA assay, using C1 protein as the antigen, in a fixed concentration coated on the plates, and the six single domain antibodies as the antibody, serially diluted. Protein concentrations in pg/ml of the single domain antibodies are indicated on the X-axis. Figure 4. This drawing is to show the ELISA assay, using R3 protein as the antigen, in a fixed concentration coated on the plates, and the six single domain antibodies as the antibody, serially diluted. Protein concentrations in pg/ml of the single domain antibodies are indicated on the X-axis. Figure 5. This drawing is to show the competition by commercial polyclonal anti-His antibody in the ELISA assay, using R3 protein as the antigen, in a fixed concentration coated on the plates, and the single domain antibody H7219 as the antibody, serially diluted. The anti His antibody were tested in two different dilutions (1:1000, and 1:5000). Protein concentrations in ptg/ml of the single domain antibodies are indicated on the X-axis.
Detailed description
The current invention describes the generation and characterization of six single domain antibodies that specifically binds to the His tag of recombinant proteins, their low-cost protection in E. coli, and their use in protein identification, quantitation, and purification. Proteins with His tag (or His-tagged proteins) are those proteins with several adjacent histidine residues coded in the protein sequence and placed at N-terminus, at C-terminus, or in middle of the protein sequence. The number of the histidine residues is usually six but can be higher or lower, with at least two histidine residues serving as metal ligands for binding. These single domain antibodies were obtained by screening of a synthetic M13 phage display library. The antigens used for the screening were two structurally unrelated proteins (Cl and R3), which both have the His tag at their C-terminus. One advantage of the single domain antibodies, in comparison with the conventional antibodies, is their low-cost production in E. coli. The obtained single domain antibodies are small and have amino acid residues ranging from 122 to 126. All of them have been highly expressed in E. coli and purified to near homogeneity by in vitro refolding. Another advantage of the single domain antibodies, in comparison with the conventional antibodies, is their structural simplicity. There is only one domain in a single domain antibody, while there are twelve domains in a conventional antibody, leading to the remarkable thermal and chemical stability of the single domain antibodies (RC Ladenson, DL Crimmins, Y Landt, JH Ladenson, Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem 2006, 78, 4501-4508). One more advantage of the single domain antibodies, in comparison with the conventional antibodies, is their simple in structure and easy for chemical and genetical modifications. The single domain antibodies can fuse genetically with a peptide sequence or a protein sequence to include extra residues for chemical modification at a specific location or gain additional functions (for example, bispecific). We provide an example of introducing two Cysteines to the C-terminus of one of the single domain antibodies. Introduction of chemically modifiable residues (thiol through cysteines, carboxyl through aspartate and glutamate, amino through lysine, or phenol through tyrosine) is important for attaching not only labels to the proteins but also the proteins to solid supports. Methods for attachments through these chemical groups (thiol, carboxyl, amino, or phenol) are well known and easily found in the literature. Affinity chromatography is the separation of a molecule or biomolecule from a mixture, based on a highly specific binding interaction. The binding of His-tagged proteins to the nickel column is one of the often-used affinity purification methods, and its popularity partially stems from their relatively low-cost and easy operation. However, His tags can exist in proteins in different conformations and has varied ability to bind to different divalent metal ions, resulting proteins in poor purities sometimes. The antibody-antigen interaction is one of the highly specific interactions, and the disclosed single domain antibodies with affinity and specificity for the His tag in recombinant proteins pave the way for affinity purification of proteins with His tag using the disclosed single domain antibodies, competing and complementing the immobilized metal-affinity chromatography (IMAC). The low-cost production of these disclosed single domain antibodies changes the affinity purification of proteins with His tag, using anti-His antibodies or anti-His single domain antibodies, from theoretically doable to economically practical. The main advantages of the present invention include: (a) They are single domain antibodies with better thermal stability, (b) They can be produced in E. coli with low cost, (c) They can be modified chemically and location-specifically as labeled proteins, (d) They have a range of different affinities that can be fine-tuned for affinity purification of His-tagged proteins. The present invention is further described in combination with specific embodiments. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1: Screening of a synthetic M13 phage display library for single domain antibodies with affinity towards proteins with His tag. A synthetic M13 phage display library was constructed by the method described in the literature with modifications (C McMahon, AS Baier, R Pascolutti, M Wegrecki, S Zheng, JX Ong, SC Erlandson, D Hilger, SGF Rasmussen, AM Ring, A Manglik, AC Kruse, Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol 2018, 25, 289-296), with NNK degenerate codons to code the sequences for the CDR1, CDR2, and CDR3 regions. Two unrelated proteins, R3 and C1, were chosen as the antigens; one is originated from a fluorescent protein, and another is from a nanobody. Their protein primary sequences and surface residues are totally different, except their common feature of a C-terminal His tag (six continuous histidine residues). The two His-tagged proteins were coated on polystyrene plates in alternate rounds of the screening, and the M13 phage library displaying single domain antibodies was added to bind to the antigens. After serval rounds of screening, the M13 phage clones with affinity towards the His-tagged proteins were enriched and selected for further characterizations.
Example 2: Expression and purification of the anti-His single domain antibodies. Six positive clones from the above screening (Fig. 1) were identified, and each of their genes was cloned into pET23a(+) vector for expression under the control of a T7 promotor in E. coli strain BL21(DE3) after induction by IPTG. Almost all antibody proteins were express highly in E. coli and found mostly in insoluble fractions as inclusion bodies. The inclusion bodies were washed with PBS buffer, and dissolved in 8 M urea. After centrifugation, the cleared 8 M urea solutions were dialyzed against PBS buffer at 4 °C. After removing precipitates, the solution contained the single domain antibodies in reasonable purity for characterization (Fig. 2).
Example 3: ELISA assays for the anti-His single domain antibodies. The six single domain antibodies were further characterized by ELISA assays. In the ELISA assay, C1 protein and R3 protein were each coated on separate plates in a fixed concentration.
The single domain antibodies were diluted in two-fold series, in sixteen different concentrations. After binding between the antigen proteins and the anti-His antibodies, the bound anti-His single domain antibodies were quantitated by using commercial anti-HA antibody and HRP-conjugated secondary antibody as reading at 450 nm absorbance. Binding curves of the six single domain antibodies when C1 or RI as the antigen are shown in Figs. 3 and 4, respectively. As controls, these proteins were tested against other proteins without His tag (such as BSA), and they did not show significant binding as indicated by readings at 450 nm.
Example 4: ELISA assays for competition of the anti-His single domain antibody with commercial polyclonal anti-His antibodies. The anti-His single domain antibody H7219 was used to compete with commercial anti His antibody (Anti-6xHis rabbit polyclonal antibody from Sangon Biotech, catalog No. D110002). The ELISA assay with R3 protein as the antigen at a fix concentration was repeated at two different dilutions (1:1000 and 1:5000) of the commercial antibody, the binding curves are shown in Fig. 5. This experiment demonstrates that the anti-His single domain antibody interacts with the antigen protein at a specific epitope, which can be blocked by the commercial anti-His antibody.
Example 5: Expression and purification of the anti-His single domain antibodies with additional sequence added to its C-terminus for chemical modification. The chemically modifiable residues on protein surface include thiol, carboxyl, amino, and phenol groups, and among these groups, thiol group usually is the fewest on surface of most proteins and the good attachment point for site-specific chemical modification (JM Kuiper, R Pluta, WH Huibers, F Fusetti, ER Geertsma, B Poolman, A method for site-specific labeling of multiple protein thiols. Protein Sci 2009, 18, 1033-1041). To illustrate the genetic modification of these single domain antibodies for easier incorporation of a label, we constructed the fusion protein by extending a short peptide sequence to the C-terminus of the protein. The sequence is two adjacent cysteine residues, linked to the C-terminal of H7219 by a GGSGG linker. The protein, named as His1, was expressed in E. coli in high yield and purified the same way as other single domain antibodies to homogeneity (Fig. 2).
Although the present invention has been described in considerable details with reference to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein.
SEQUENCE LISTING 19 Nov 2020
<110> Next Medicine Co., Ltd.
<120> Anti-His single domain antibodies and use thereof
<130> temp
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<400> 16
Ile Ser Leu Glu Ala Glu Met Gly 1 5
<210> 17 <211> 15 <212> PRT <213> Artificial Sequence
<220> <223> synthetic
<400> 17
Ile Ser Arg Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
<210> 18 <211> 20
<212> PRT 19 Nov 2020
<213> Artificial Sequence
<220> <223> synthetic
<400> 18
Ala Val Pro His Ala Ile His Ser Pro Ala Arg Ile Ile Tyr Phe Leu 2020103565
1 5 10 15
His Leu Met Tyr 20
<210> 19 <211> 122 <212> PRT <213> Artificial Sequence
<220> <223> synthetic
<400> 19
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Asn Phe Asp 20 25 30
Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45
Ala Thr Ile Asn Ser Gly Ala Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Ala Ser His Tyr Tyr His Leu Ile Leu Ser Phe Gln Leu Ser Tyr Trp 100 105 110 2020103565
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
<210> 20 <211> 122 <212> PRT <213> Artificial Sequence
<220> <223> syntehtic
<400> 20
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Tyr Thr Arg 20 25 30
Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45
Ala Ala Ile Asn Tyr Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 19 Nov 2020
85 90 95
Val Leu Ser Ser Lys Ser His Lys Tyr Ile Tyr Arg Phe Tyr Tyr Trp 100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 2020103565
115 120
<210> 21 <211> 125 <212> PRT <213> Artificial Sequence
<220> <223> synthetic
<400> 21
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Leu Arg His 20 25 30
Tyr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45
Ala Ala Ile Ser Ser Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Val Glu Leu Phe Pro Arg Pro Tyr Arg Tyr Leu Gly Met Ile Asn Leu 100 105 110
Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 2020103565
<210> 22 <211> 126 <212> PRT <213> Artificial Sequence
<220> <223> synthetic
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Leu Glu Ala 20 25 30
Glu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45
Ala Thr Ile Ser Pro Gly Ser Ile Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Val Leu Gly Pro Ala Leu Met Met Ala Ile Asp Asp Arg Ala Met Asp 19 Nov 2020
100 105 110
Phe Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
<210> 23 2020103565
<211> 126 <212> PRT <213> Artificial Sequence
<220> <223> synthetic
<400> 23
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Thr Ser Ala 20 25 30
Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45
Ala Thr Ile Asn Ala Gly Thr Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Val Gly Trp Val Trp Gly Ala Pro Trp Leu His Thr Ile Ala Ala Val 100 105 110
Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
<210> 24 <211> 126 <212> PRT 2020103565
<213> Artificial Sequence
<220> <223> synthetic
<400> 24
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Leu Glu Ala 20 25 30
Glu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45
Ala Gly Ile Ser Arg Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Val Pro His Ala Ile His Ser Pro Ala Arg Ile Ile Tyr Phe Leu His 100 105 110
Leu Met Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 19 Nov 2020
115 120 125

Claims (6)

EDITORIAL NOTE 2020103565 There is one page of claims only Claims
1. A single domain antibody that specifically binds proteins with His-tag, which has three complementary determining regions (CDR1, CDR2, and CDR3), contains: (a) a CDR1, CDR2, CDR3 combination sequence defined by i. SEQ ID NOS: 1, 2, and 3, as CDR1, CDR2, and CDR3, respectively, ii. SEQ ID NOS: 4, 5, and 6, as CDR1, CDR2, and CDR3, respectively, iii. SEQ ID NOS: 7, 8, and 9, as CDR1, CDR2, and CDR3, respectively, iv. SEQ ID NOS: 10, 11, and 12, as CDR1, CDR2, and CDR3, respectively, v. SEQ ID NOS: 13, 14, and 15, as CDR1, CDR2, and CDR3, respectively, or vi. SEQ ID NOS: 16, 17, and 18, as CDR1, CDR2, and CDR3, respectively; or (b) a sequence that has at least 80%, preferably 90%, more preferably 95% sequence identity with one of the above CDR1, CDR2, CDR3 combination sequences.
2. A single domain antibody that specifically binds proteins with His-tag, contains: (a) a polypeptide sequence defined by SEQ ID NOS: 19, 20, 21, 22, 23, or 24; or (b) a sequence that has at least 80%, preferably 90%, more preferably 95% sequence identity with the above polypeptide sequence.
3. A fusion protein that contains the single domain antibody of claims 1 or 2, and the fusion part comprises: (a) short peptide sequences, (b) binding proteins, (c) fluorescent proteins, or (d) enzymes such as luciferase, alkaline phosphatase, horseradish peroxidase, and sortase.
4. A protein of claims 1, 2 or 3 that is modified by attaching labels covalently or non covalently, and the label comprises: (a) dyes, (b) fluorescent compounds, (c) luminogenic compounds, (d) gold nanoparticles, (e) biotin, and biotin analogs and derivatives, or (f) enzymes such as luciferase, alkaline phosphatase, horseradish peroxidase.
5. A protein of claims 1, 2 or 3 that is attached covalently to a solid support comprising polymer membranes, chromatographic resins, polymer and magnetic beads.
6. Use of the proteins of claims 3, 4 or 5 for the purpose of identification, quantitation, and purification of proteins with His-tag.
Figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
AU2020103565A 2020-11-19 2020-11-19 Anti-His single domain antibodies and use thereof Ceased AU2020103565A4 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116554338A (en) * 2023-03-01 2023-08-08 湖南诺合新生物科技有限公司 His tag-resistant monoclonal antibody and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116554338A (en) * 2023-03-01 2023-08-08 湖南诺合新生物科技有限公司 His tag-resistant monoclonal antibody and application thereof
CN116554338B (en) * 2023-03-01 2024-03-08 湖南诺合新生物科技有限公司 His tag-resistant monoclonal antibody and application thereof

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