WO2021249433A1 - 蛋白固定化用载体及其制备方法 - Google Patents

蛋白固定化用载体及其制备方法 Download PDF

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WO2021249433A1
WO2021249433A1 PCT/CN2021/099144 CN2021099144W WO2021249433A1 WO 2021249433 A1 WO2021249433 A1 WO 2021249433A1 CN 2021099144 W CN2021099144 W CN 2021099144W WO 2021249433 A1 WO2021249433 A1 WO 2021249433A1
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carrier
peptide
spycatcher
spytag
epoxy
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PCT/CN2021/099144
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English (en)
French (fr)
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林章凛
叶燕锐
杨晓锋
林巧
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华南理工大学
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Priority to CN202180041920.6A priority Critical patent/CN115698281A/zh
Publication of WO2021249433A1 publication Critical patent/WO2021249433A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent

Definitions

  • the present invention relates to the field of bioengineering, in particular to the field of protein immobilization such as enzyme immobilization, and in particular to a protein immobilization method, a modified carrier for immobilizing a protein, and a modified carrier to which the protein is immobilized.
  • immobilized protein such as enzyme
  • free protein immobilized protein
  • Polymerized epoxy carriers (such as polypropylene and polystyrene) have a wide range of applications in the immobilization of industrial enzymes due to their high mechanical stability [Boller, Thomas, Christian Meier, and Stefan Menzler.”Eupergit oxirane acrylic beads: how to make enzymes fit for biocatalysis. "Organic Process Research & Development 6.4(2002):509-519; Mateo,Cesar,et al.”Immobilization of enzymes on heterofunctional epoxy supports.”Nature Protocols 2.5(2007):1022;McAuliffe:1022; ,Joseph C. "Industrial enzymes and biocatalysis.” Handbook of industrial chemistry and biotechnology. Springer, Boston, MA, 2012.1183-1227].
  • Epoxy activated carrier (hereinafter referred to as epoxy carrier) can pass through the epoxy group on the carrier and a variety of nucleophilic groups on the surface of the protein (such as lysine, cysteine, histidine, tyrosine, etc.) , So it can achieve multi-point covalent immobilization of protein.
  • the types and properties of epoxy carriers have a great influence on the results of protein immobilization. For the same enzyme, using different epoxy carriers, the immobilization efficiency and activity recovery rate are often very different [Seip,John E.,et al .”Glyoxylic acid production using immobilized glycolate oxidase and catalase.” Bioorganic & medical chemistry 2.6 (1994): 371-378].
  • the directional immobilization of the protein has a uniform orientation and can maintain the activity of the protein to the maximum. Therefore, the directional immobilization of the protein has a wide range of applications in the fields of industrial biocatalysis, clinical diagnosis, and protein interaction analysis [Wong, Lu Shin, Farid Khan, and Jason Micklefield.”Selective covalent protein immobilization: strategies and applications.”Chemical reviews 109.9(2009):4025-4053.; Wong, Lu Shin, Farid Khan, and Jason Micklefield.”Selective covalent protein immobilization: strategies and applications.
  • the protein can be linked to the N-terminus of the oligoglycine to achieve targeted protein immobilization; formylglycine is produced After the enzyme (FGE) modifies the cysteine residue (C) on the CXPXR, it can be linked to the amino group of the carrier through the Schiff base reaction to achieve the targeted immobilization of the protein [Popp, Maximilian W., et al.”Sortagging: a versatile method for protein labeling.
  • Sortase A-mediated immobilization rate is low, generally around 30% [Hata, Yuto, et al.”C-Terminal-oriented Immobilization of Enzymes Using Sortase A-mediated Technique.”Macromolecular bioscience 15.10(2015):1375-1380; Ito,Takaomi,et al.”Highly oriented recombinant glycosyltransferases: site-specific immobilization of unstable membrane proteins by using Staphylococcus 49 aureus sortase 2604 (2010) A.” -2614].
  • a SpyCatcher peptide modified carrier wherein the carrier, when not modified by the SpyCatcher peptide, contains a group capable of reacting with an amino group (NH 2 ), wherein the SpyCatcher peptide reacts with the amino group through the amino group.
  • the reaction of the group is covalently attached to the carrier, and the SpyCatcher peptide connected to the carrier can form an isopeptide bond with the Spytag peptide.
  • the group is selected from epoxy, aldehyde, imide, cyanate, imine carbonate, and hydrazide.
  • the SpyCatcher peptide preferably comprises the amino acid sequence shown in SEQ ID NO:21. In one embodiment, the SpyCatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 1, 20, 22 or 23.
  • the carrier is further connected to a fusion protein comprising a SpyTag peptide and a target protein, wherein the SpyCatcher peptide forms an isopeptide bond with the SpyTag peptide, and the isopeptide bond is formed by the SpyCatcher peptide corresponding to The Lys residue of Lys at position 56 of the amino acid sequence shown in SEQ ID NO: 1 is formed with the Asp residue of Asp at position 7 of SEQ ID NO: 2 on the SpyTag peptide.
  • the protein of interest is an enzyme.
  • the SpyTag peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 11-15, and 28.
  • the protein of interest is an enzyme, preferably selected from the group consisting of glutaryl-7-aminocephalosporanic acid acyltransferase, glucose isomerase, nitrile hydratase, penicillin amidase, aspartase, fumaric acid Enzymes, aminoacylase, lactase, aspartate- ⁇ -decarboxylase and cephalosporin amidase.
  • the carrier is made of an inorganic material, an organic material, or a composite material of an inorganic material and an organic material, wherein the inorganic material includes, but is not limited to, silica, metal oxide, clay material, and the organic material
  • the inorganic material includes, but is not limited to, silica, metal oxide, clay material, and the organic material
  • the materials include but are not limited to agarose, chitosan, alginate, gelatin, polyacrylic acid, polyacrylate, polystyrene, polyamide, polyacrylonitrile, and preferably the carrier is epoxy resin or amino resin.
  • a SpyCatcher peptide modified epoxy resin carrier wherein the SpyCatcher peptide is covalently attached to the carrier through the reaction of an amino group and an epoxy group, preferably the epoxy resin carrier Made of polyacrylate.
  • the particle size of the epoxy resin is 100-1000 ⁇ m, and/or the epoxy group content of the epoxy resin is 50-800 ⁇ mol/g wet.
  • a method for preparing a SpyCatcher peptide-modified carrier which includes:
  • a method for immobilizing proteins including:
  • step (3) Contact the carrier described in step (1) with the fusion protein described in step (2) under conditions that allow the SpyCatcher peptide and the SpyTag peptide to form isopeptide bonds, optionally, after the contact , Washing with washing buffer to remove unimmobilized fusion protein; and
  • the method can be used to obtain a homogeneous enzyme immobilization carrier.
  • the SpyTag peptide comprises the amino acid sequence shown in SEQ ID NO: 21, 22 or 23. In one embodiment, the SpyTag peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 11-15, and 28.
  • step (1) includes incubating the SpyCatcher peptide with a carrier that has not been modified by the SpyCatcher peptide to obtain a SpyCatcher peptide-modified carrier; and optionally, treating the SpyCatcher peptide-modified carrier with a blocking buffer.
  • the protein of interest is an enzyme, preferably selected from the group consisting of glutaryl-7-aminocephalosporanic acid acyltransferase, glucose isomerase, nitrile hydratase, penicillin amidase, aspartase, fumaric acid Enzymes, aminoacylase, lactase, aspartate- ⁇ -decarboxylase and cephalosporin amidase.
  • the carrier is selected from epoxy resins and amino resins, and is preferably a carrier made of inorganic materials, organic materials, or a composite of inorganic and organic materials, wherein the inorganic materials include but are not limited to two Silica, metal oxides, clay materials, or carriers made of organic materials, including but not limited to agarose, chitosan, alginate, gelatin, polyacrylic acid, polyacrylate, polyphenylene Ethylene, polyamide, polyacrylonitrile.
  • a kit which comprises a carrier, a SpyCatcher peptide, and optionally a fusion protein comprising a SpyTag peptide and a target protein, wherein the carrier and the SpyCatcher peptide exist alone or in the form of the SpyCatcher peptide described in the first aspect.
  • Peptide-modified carrier exists.
  • the protein of interest is an enzyme.
  • Figure 1 Schematic diagram of protein-specific immobilization method based on SpyTag-SpyCatcher reaction.
  • Protein expression plasmid map a) SpyCatcher expression plasmid map; b) SpyTag-RFP expression plasmid map; c) SpyTag-GA expression plasmid map.
  • FIG. 3 SDS-PAGE image of SpyCatcher, SpyTag-RFP and SpyTag-GA expression and purification.
  • Lanes 1-5 Protein quantification standards containing bovine serum protein BSA, and the loading concentrations are 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • Lanes 6, 7, and 8 are the fusion proteins SpyTag-GA, SpyCatcher and SpyTag-RFP, respectively.
  • Figure 4 SDS-PAGE of protein before and after SpyCatcher immobilized on epoxy carrier.
  • Lanes 1-4 SpyCatcher supernatants with 5, 10, 20, and 40 mg/g carrier before fixation
  • Lanes 5-8 SpyCatcher supernatants with 5, 10, 20, 40 mg/g carrier after fixation
  • Lane 9-13 Protein quantification standard containing bovine serum albumin BSA, the loading concentration is 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • FIG. 5 Immobilized Spy-RFP.
  • a) SDS-PAGE images of purified SpyTag-RFP before and after fixation Lane 1: SpyTag-RFP supernatant before fixation; Lane 2: Supernatant after SpyTag-RFP fixation with the method of the present invention; Lane 3: Blocking control fixation of SpyTag-RFP Lane 4: The supernatant after SpyTag-RFP was immobilized by epoxy immobilization method; Lane 5-9: The protein quantitative standard containing bovine serum protein BSA, the loading concentration was 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • Figure 6 Immobilization of SpyTag-RFP directly from the supernatant of E. coli cell lysate. a) SDS-PAGE images of E. coli cell lysate overexpressing SpyTag-RFP before and after fixation.
  • Lane 1 SpyTag-RFP cell lysis supernatant before fixation
  • Lane 2 Cell lysis after SpyTag-RFP fixation with the method of the present invention Supernatant
  • Lane 3 Blocking control cell lysis supernatant after fixing SpyTag-RFP
  • Lane 4 Epoxy method of cell lysis supernatant after fixing SpyTag-RFP
  • Lane 5-9 Containing bovine serum albumin BSA
  • the loading concentrations were 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • coli cell lysate supernatant overexpressing SpyTag-RFP before and after fixation lane 1: SpyTag-RFP cell lysis supernatant before fixation; Lane 2: cell lysis after SpyTag-RFP fixation with the method of the present invention Clear liquid; Lane 3: Blocking control cell lysis supernatant after fixing SpyTag-RFP; Lane 4: Epoxy immobilization method cell lysis supernatant after fixing SpyTag-RFP; Lane 5-9: Containing bovine serum albumin BSA The protein quantification standards of, the sample concentration is 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • FIG. 7 SDS-PAGE image of the supernatant before and after the immobilization of purified SpyTag-GA.
  • Lane 1 SpyTag-GA supernatant before fixation
  • Lane 2 Supernatant after SpyTag-GA fixation by the method of the present invention
  • Lane 3 Blocking control supernatant after fixation of SpyTag-GA
  • Lane 4 Epoxy immobilization Methods The supernatant after fixing SpyTag-GA
  • lanes 5-9 protein quantification standard containing bovine serum protein BSA, the loading concentration is 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • FIG. 8 SDS-PAGE image of SpyTag-GA immobilized directly from the supernatant of E. coli cell lysate.
  • Lane 1 SpyTag-GA cell lysis supernatant before fixation
  • Lane 2 Cell lysis supernatant after SpyTag-GA immobilization in the method of the present invention
  • Lane 3 Blocking control cell lysis supernatant after SpyTag-GA immobilization
  • Lane 4 The cell lysis supernatant after fixing SpyTag-GA by epoxy immobilization method
  • Lanes 5-9 Protein quantification standard containing bovine serum albumin BSA, the loading concentration is 31.25 ⁇ g/mL, 62.5 ⁇ g/mL, 125 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL.
  • Figure 9 Comparison of the immobilization efficiency of the SpyCatcher modified carrier and the epoxy carrier immobilized protein in the method of the present invention.
  • Figure 10 Comparison of the immobilization efficiency of the method of the present invention and the epoxy method using the same carrier to immobilize proteins. a) LX1000-EP; b) LX-107S; c) LX-103B; d) HFA001; e) ECR8204; f) ECR8285.
  • Figure 11 Comparison of the activity recovery rate of the SpyCatcher modified carrier and the epoxy carrier immobilized protein in the method of the present invention.
  • Figure 12 Comparison of the activity recovery rate of the protein immobilized with the same carrier in the method of the present invention and the epoxy method. a) LX1000-EP; b) LX-107S; c) LX-103B; d) HFA001; e) ECR8204; f) ECR8285.
  • Figure 13 Comparison of relative activity of SpyCatcher modified carrier and epoxy carrier immobilized protein in the method of the present invention.
  • Figure 14 Comparison of relative activity of protein immobilized with the same carrier in the method of the present invention and the epoxy method. a) LX1000-EP; b) LX-107S; c) LX-103B; d) HFA001; e) ECR8204; f) ECR8285.
  • the present invention provides a protein immobilization method to solve the technical problem of protein immobilization, which is sensitive to the types of proteins and carriers, and requires trial and error experiments of immobilization and carrier screening.
  • the present disclosure provides a SpyCatcher peptide modified carrier, wherein the carrier, when not modified by the SpyCatcher peptide, contains a group capable of reacting with an amino group (NH 2 ), wherein the SpyCatcher peptide passes through The amino group reacts with the group to be covalently attached to the carrier, and the SpyCatcher peptide connected to the carrier can form an isopeptide bond with the Spytag peptide.
  • the carrier when not modified by the SpyCatcher peptide, contains a group capable of reacting with an amino group (NH 2 ), wherein the SpyCatcher peptide passes through The amino group reacts with the group to be covalently attached to the carrier, and the SpyCatcher peptide connected to the carrier can form an isopeptide bond with the Spytag peptide.
  • the carrier is any carrier that can be used to immobilize proteins, as long as it contains a group capable of reacting with an amino group (NH 2 ).
  • the carrier may be made of various materials, including but not limited to inorganic materials, organic materials, or composite materials of inorganic materials and organic materials.
  • the inorganic materials include, but are not limited to, diatomaceous earth, kaolinite, silica gel, porous glass, activated carbon, calcium carbonate, ceramics, silica, metal oxides, and clay materials.
  • the organic materials include, but are not limited to, agarose, chitosan, alginate, gelatin, polyacrylic acid, polyacrylate, polymethacrylic acid, polymethacrylate, polystyrene, poly Amide, polyacrylonitrile.
  • the carrier is an epoxy resin carrier or an amino resin carrier.
  • the carrier is a carrier made of polyacrylic acid (ester), especially an epoxy resin carrier.
  • a group capable of reacting with an amino group refers to a group capable of reacting with an amino group without using or using an activator. It is known in the art how to activate a group to react with an amino group, for example, see Hermanson, Greg T. Bioconjugate techniques. Academic press, 2013.
  • the amino group may be the amino group at the N-terminus of the peptide, or the amino group on the side chain of an amino acid residue, such as the ⁇ -amino group of Lys.
  • the SpyCatcher peptide is attached to the carrier through the reaction of one or more amino groups with the group.
  • the SpyCatcher peptide is attached to the carrier through the reaction of an amino group with the group. In one embodiment, the SpyCatcher peptide is attached to the carrier through the reaction of the N-terminal amino group with the group. In another embodiment, the SpyCatcher peptide is attached to the carrier through the reaction of an epsilon-amino group of Lys with the group.
  • the SpyCatcher peptide is attached to the carrier at multiple points through the reaction of multiple (ie, two or more) amino groups with the group.
  • the two or more (for example, 2, 3, 4, 5, 6, 7, 8, 9) amino groups may be N-terminal amino groups and one or more ⁇ -amino groups of Lys, or two or more The ⁇ -amino group of a Lys.
  • the group capable of reacting with an amino group is selected from epoxy group, aldehyde group, imide, cyanate ester, imine carbonate and hydrazide group.
  • Asp117 and Lys31 residues are very important for the Spy reaction. For example, if the ⁇ -NH 2 of Lys31 reacts with other groups, the Spy reaction will not occur. See, for example, Science, 2007, 318, 1625-1628] [PNAS, 2012; 109:690; Angew. Chem. Int. Ed. 2010, 49, 8421-8425, JACS. 2011, 133, 478-485.
  • Spy chemistry has been extensively studied in protein labeling, protein binding, protein topology and protein materials (PNAS, 2012; 109:690; Bioconjugate Chem. 2017, 28: 1544-1551; J. Am. Chem. Soc. 2013, 135:13988-13997,Angew.Chem.Int.Ed.2019,58,11097-11104; Angew.Chem.Int.Ed.2014,53:6101-6104; PNAS,2014,111,31,11269- 11274, Matter 1, 1-17).
  • SpyCatcher has high specificity, SpyTag (13 amino acids) and SpyCatcher (115 amino acids) are relatively small, and SpyCatcher can be obtained by conventional recombinant expression technology (for example, using E. coli as the expression host, with every L The fermentation broth can get 110-150mg Histag purified SpyCatcher protein).
  • the present disclosure is based on Spy chemistry and provides a protein immobilization method: including covalently connecting the SpyCatcher and the carrier group through a covalent bond to obtain a SpyCatcher modified carrier, and then using Spy chemistry to fix the fusion protein of the SpyTag and the target protein to SpyCatcher modified vector.
  • the inventors unexpectedly discovered that after reacting the SpyCatcher peptide with the carrier, Lys31 can still form an isopeptide bond with Asp117, resulting in a Spy reaction.
  • protein protein
  • peptide polypeptide
  • amino acid sequence refers to polymers of any length, for example, two or more amino acid residues.
  • the term also includes amino acid polymers modified naturally or artificially; for example, formation of disulfide bonds, glycosylation, esterification, acetylation, phosphorylation, or any other manipulations and modifications, such as labeling or biologically active groups. Sub-conjugation. This article uses conventional one-letter or three-letter amino acid residue codes.
  • amino acid refers to natural and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids.
  • Natural amino acids are those encoded by the genetic code and those amino acids that are later modified, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids (that is, ⁇ -carbon combined with hydrogen, carboxyl, amino, and R group).
  • Amino acid mimics refer to chemical compounds whose structure differs from the general chemical structure of amino acids but functions in a manner similar to natural amino acids.
  • the SpyCatcher peptide is any peptide or variant thereof capable of reacting with the SpyTag peptide or variants thereof to form isopeptide bond linkages (Spy reaction).
  • SpyCatcher peptides and variants thereof are known in the art, for example, see Zakeri, B., Fierer, JO, Celik, E., Chittock, EC, Schwarz-Linek, U., Moy, VT, & Howarth, M.
  • the SpyCatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence with one or more amino acid substitutions.
  • the substitution is an amino acid substitution at one or more of the following positions in SEQ ID NO: 21: 15, 40, 67, 69, 75, 81, 83, 86, 91.
  • the substitution is one or more substitutions selected from the following: K15R, Q40H, A67P, T69E, Q75D, N81D, K83E, K86E, I91T.
  • the SpyCatcher peptide includes the amino acid sequence shown in SEQ ID NO: 21, for example, the amino acid sequence shown in SEQ ID NO: 1 or 20.
  • the SpyCatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 20 and has amino acid substitutions at one or more of the following positions in the amino acid sequence shown in SEQ ID NO: 20: 2, 9, 13, 19 , 37, 62, 89, 91, 97, 103, 105, 108, and 113.
  • the one or more amino acid substitutions are selected from the following amino acid substitutions at one or more positions of the amino acid sequence shown in SEQ ID NO: 20: D2T, S9G, Q13P, I19T, K37R, Q62H , A89P, T91E, Q97D, N103D, K105E, K108E, I113T.
  • the one or more amino acid substitutions are one or more amino acid substitutions selected from the following amino acid sequence shown in SEQ ID NO: 20: D2T, S9G, Q13P, I19T, K37R, Q62H, K105E , I113T.
  • the SpyCatcher peptide comprises the amino acid sequence shown in SEQ ID NO: 22 or 23.
  • the SpyCatcher peptide with one or more amino acid substitutions according to the present invention may comprise at least 80%, at least 85%, or at least the amino acid sequence shown in SEQ ID NO: 1, 20, 21, 22 or 23 An amino acid sequence of 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity.
  • Sequence identity can be determined by a commercially available computer program, which uses any suitable algorithm to calculate the percent identity between two or more sequences, such as using default parameters.
  • a typical example of such a computer program is CLUSTAL.
  • the BLAST algorithm is applied, and the parameters are set to default values.
  • NCBI National Center for Biotechnology Information
  • the carrier is an epoxy carrier or an amino carrier, that is, epoxy groups or amino groups are present on the carrier, such as epoxy resins and amino resins. Due to the chemical activity of epoxy groups, a variety of compounds containing active hydrogen can be used to open the ring. Among covalent carriers, epoxy carriers are almost ideal substrates, which can easily immobilize proteins on a laboratory and industrial scale. Under very mild experimental conditions (such as pH 7.0), the epoxy carrier reacts with the protein.
  • the carrier is a carrier made of an inorganic material, an organic material, or a composite material of an inorganic material and an organic material, where the inorganic material includes, but is not limited to, silica, metal oxide, and clay material, for example.
  • the organic materials include, but are not limited to, agarose, chitosan, alginate, gelatin, polyacrylic acid, polyacrylate, polymethacrylic acid, polymethacrylate, polystyrene, polyamide, polypropylene Nitrile.
  • epoxy resin refers to a resin containing epoxy groups
  • amino resin refers to a resin containing amino groups, wherein the epoxy groups and amino groups may be epoxy groups and amino groups obtained by modification on the resin.
  • epoxy resin refers to a polymer containing epoxy groups, and may include, for example, epoxy resins, amino epoxy resins, carboxy epoxy resins, and mercapto-disulfide epoxy resins.
  • Epoxy resins include, but are not limited to, Lifetech TM ECR8285, ECR8204, ECR8209, LX1000EA, LX1000EP, LX103B, EP200, LX1000HFA, HFA001, LX107S, LX1000SW, LX1000SD, C, C250L, FP-EC3, EC-EP/M, EC-Ep, ES1, ES103, ES105, ES108 and ES109.
  • the particle size of the epoxy resin is about 5-2000 ⁇ m, such as about 10-2000, 20-2000, 30-2000, 40-2000, 50-2000, 50-1900, 50-1800, 50 -1700, 50-1600, 50-1500, 50-1400, 50-1300, 50-1200, 50-1100, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500 , 50-450, 50-400, 50-350, 100-450, 100-400, 100-350, 100-300, 150-450, 150-400, 150-350, 150-300 ⁇ m, such as about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,
  • the epoxy group content of the epoxy resin is less than 1000 ⁇ mol/g wet, such as about 10-1000 ⁇ mol/g wet, for example, about 10-900, 10-800, 10-700, 10-600, 10 -500, 10-400, 10-300, 10-200, 10-150, 10-100, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300 , 20-200, 20-150, 20-100, 30-900, 30-800, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-150, 30 -100, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-150, 40-100, 50-900, 50-800 , 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-150, 50-100, 50-90, 50-80, 70-90, 80-100, 75 -95, 390-520, 585-7
  • the epoxy resin is hydrophilic.
  • the epoxy resin is made of polyacrylate, such as polymethyl acrylate, ethyl acrylate, propyl ester, butyl ester, isobutyl ester, tert-butyl ester, and the like.
  • the epoxy resin is made of polymethyl acrylate.
  • the epoxy resin is made of polyethyl acrylate.
  • the epoxy resin is made of polypropyl acrylate.
  • the epoxy resin is made of polybutyl acrylate.
  • the epoxy resin is made of polyisobutyl acrylate.
  • the epoxy resin is made of poly-tert-butyl acrylate.
  • the group attached to the Spycatcher peptide on the epoxy resin is an epoxy group.
  • a SpyCatcher peptide modified epoxy resin carrier as described herein, wherein the SpyCatcher peptide is covalently attached to the carrier by reacting an amino group with an epoxy group of the resin.
  • the epoxy resin carrier is made of polyacrylate. More preferably, the particle size of the epoxy resin is about 100-350 ⁇ m, and/or the epoxy content of the epoxy resin is about 50-100 ⁇ mol/g wet.
  • amino (NH 2 ) resin refers to a polymer containing amino groups, and may include, for example, polyacrylates containing amino groups, polymethacrylates containing amino groups, polystyrenes containing amino groups, and the like.
  • amino resins include but are not limited to LX-1000EA, LX-1000HA, ECR8305, ECR8309, ECR8315, ECR8404, ECR8409, ECR8415.
  • a method for preparing a SpyCatcher peptide-modified vector including:
  • the carrier, SpyCatcher peptide, etc. are as described above.
  • the unmodified vectors described herein can be obtained by any means known in the art.
  • the carrier Before being contacted with the SpyCatcher peptide, the carrier can be activated with an activator, or can be directly contacted with the SpyCatcher peptide (without using an activator).
  • the reaction conditions are known in the art or can be easily determined according to techniques known in the art.
  • a liquid environment refers to a liquid environment suitable for the SpyCatcher peptide to be covalently attached to the peptide by reacting the amino group (such as the N-terminal and side chain amino group, or the side chain amino group) on the peptide with the group capable of reacting with the amino group.
  • the liquid environment of the carrier for example, PBS buffer (such as 0.1M, pH 7.0).
  • PBS buffer such as 0.1M, pH 7.0.
  • activation refers to the treatment of the group so that it can react with the amino group on the SpyCatcher peptide, for example, glutaraldehyde is used as an activator to activate the amino group on the carrier.
  • glutaraldehyde is used as an activator to activate the amino group on the carrier.
  • step (2) of the method of the second aspect includes incubating the SpyCatcher peptide with an unmodified carrier, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. , 11, 12, 24, 36, and 48 hours to obtain the SpyCatcher peptide-modified carrier; and optionally, treat the SpyCatcher peptide-modified carrier with a blocking buffer, for example, at least about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 24, 36, 48 hours.
  • the incubation is centrifuged at about 20-50 rpm, such as about 25-40, 25-35, 25-30 rpm. After the blocking is completed, the modified vector can be separated, for example by centrifugation to remove the blocking buffer.
  • the ratio of SpyCatcher peptide to unmodified carrier used for incubation is 10-40 mg peptide/g carrier, such as 10, 15, 20, 25, 30, 35, 40 mg peptide/g carrier, particularly preferably 20 mg Peptide/g carrier.
  • the method step (2) of the second aspect can be carried out at any suitable temperature and pH.
  • the temperature may be about 20-37°C, such as about 20-35°C, 25-37°C, 25-35°C, 20-30°C, 25-35°C, especially about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37°C.
  • the pH value can be any suitable pH, for example about 4.0-10.0, such as about 5.0-9.0, 6.0-8.0 or 6.0-7.0, especially about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0.
  • blocking refers to the reaction of the remaining groups that have not reacted with SpyCatcher, such as epoxy groups.
  • the protein reacts.
  • the blocking buffer described herein is a blocking buffer commonly used in the art. Suitable blocking buffers for the groups described herein are known in the art (for example, see Hermanson, Greg T. Bioconjugate technologies. Academic press, 2013; Immobilization of enzymes and cells. Humana Press, 2006).
  • the blocking buffer described herein is an epoxy group blocking buffer commonly used in the art, such as glycine, ethanolamine, bovine serum albumin and the like.
  • the blocking buffer includes but is not limited to: glycine, pH 8.5; ethanolamine, pH 8.5.
  • the blocking buffer is 3M glycine, pH 8.5.
  • the separation of the SpyCatcher peptide-modified carrier described in step (3) of the method of the second aspect can be separated by any suitable technique known in the art, such as centrifugation.
  • the SpyCatcher peptide-modified carrier described herein or obtained according to the method described herein is connected to a fusion protein comprising a SpyTag peptide and a target protein, wherein the SpyCatcher peptide is an isopeptide formed from the SpyTag peptide Bond (Spy reaction) to connect the target protein to the SpyCatcher peptide-modified carrier.
  • the SpyTag peptide is also any peptide known in the art that can undergo Spy reaction.
  • Various techniques and methods for producing fusion proteins are known in the art.
  • the SpyTag peptide can be located at any position of the N-terminal, C-terminal or middle of the fusion protein, as long as the obtained fusion protein has the required functional activity.
  • a person skilled in the art can easily detect the functional activity of a protein according to the technical knowledge in the art, which is within the technical ability of the person skilled in the art.
  • the SpyTag peptide described herein comprises the amino acid sequence shown in any one of SEQ ID NO: 2, 11, 12, 13, 14, 15 or 28.
  • the peptide SpyTag is located at the N-terminus or C-terminus of the fusion protein.
  • the SpyTag peptide and the target protein in the fusion protein of the present invention can be connected by a linker, such as SpyTag-linker-target protein or target protein-linker-SpyTag.
  • the linker is a peptide or other molecule that connects the SpyTag peptide and the target protein.
  • the connection can be connected by any method known in the art to connect the two parts, as long as the linker part does not significantly hinder the desired functional activity of the protein in the fusion protein and/or does not significantly hinder the Spy reaction between the SpyTag peptide and the SpyCatcher peptide That's it.
  • Those skilled in the art can easily determine and select a suitable linker based on the technical knowledge in the art.
  • the fusion protein of the SpyTag peptide and the target protein of the present invention includes the SpyTag peptide and the target protein, and may also include a polypeptide linker and an affinity tag.
  • the polypeptide linker is located between the SpyTag and the target protein.
  • affinity tag refers to an affinity tag added to the N-terminus or C-terminus of the fusion protein to facilitate subsequent purification of the target protein.
  • the fusion protein of the SpyTag and the target protein can be a purified fusion protein or an unpurified fusion protein, such as a cell lysate.
  • the amino acid sequence of the SpyTag is shown in SEQ ID NO: 2.
  • the SpyTag peptide can be at the C-terminus or N-terminus of the target protein.
  • the linker is preferably not less than 9 amino acid residues in length, for example, 9, 11, 13, 15 amino acid residues or longer. Any linker known to those skilled in the art can be used in the present invention.
  • the target protein linked to the SpyTag can be any protein desired to be immobilized on the carrier, including, for example, but not limited to, enzymes, cofactors, chaperone proteins, and the like.
  • the protein of interest is an enzyme, such as any enzyme known in the art that can be immobilized on the carrier described herein, and the enzyme can be from any suitable source, which can be isolated from a natural source For example, bacterial or artificially synthesized, for example, expressed by recombinant technology.
  • the enzyme is selected from glucose isomerase (EC 5.3.1.5), nitrile hydratase (EC 4.2.1.84), penicillin amidase (EC 3.5.1.11), aspartase (EC 4.3. .1.1), fumarase (EC 4.2.1.2), aminoacylase (EC 3.5.1.14), lactase (EC 3.2.1.108), aspartate- ⁇ -decarboxylase (EC 4.1.1.12) and cephalosporin Bacteriocin amidase (EC 3.5.1.11).
  • the present disclosure provides a SpyCatcher peptide modified epoxy resin carrier, wherein the SpyCatcher peptide is covalently attached to the carrier by reacting the amino group with the epoxy group of the carrier.
  • the SpyCatcher peptide has the amino acid sequence shown in SEQ ID NO:1.
  • the epoxy resin is made of polyacrylate.
  • the particle size of the epoxy resin is about 100-350 ⁇ m, and/or the epoxy group content of the epoxy resin is about 50-100 ⁇ mol/g wet.
  • the present disclosure provides an epoxy resin carrier immobilized with a protein, the epoxy resin is connected to the SpyCatcher peptide having the amino acid sequence shown in SEQ ID NO: 1, and the protein contains SEQ ID NO: : A fusion protein of the SpyTag peptide with the amino acid sequence shown in 2 and the target protein.
  • the epoxy resin is made of polyacrylate.
  • the present disclosure provides a method for immobilizing proteins, including:
  • step (3) Contact the carrier described in step (1) with the fusion protein described in step (2) under conditions that allow the SpyCatcher peptide and the SpyTag peptide to form isopeptide bonds, optionally, after the contact , Washing with washing buffer to remove unimmobilized fusion protein; and
  • the carrier, SpyCatcher peptide, SpyTag peptide, protein, etc. are as described above.
  • the method step (1) of the third aspect includes incubating the SpyCatcher peptide with an unmodified carrier, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. , 11, 12, 24, 36, and 48 hours to obtain the SpyCatcher peptide-modified carrier; and optionally, treat the SpyCatcher peptide-modified carrier with a blocking buffer, for example, at least about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 24, 36, 48 hours.
  • the incubation is centrifuged at about 20-50 rpm, such as about 25-40, 25-35, 25-30 rpm. After the blocking is completed, the modified vector can be separated, for example by centrifugation to remove the blocking buffer.
  • the ratio of SpyCatcher peptide to unmodified carrier used for incubation is 10-40 mg peptide/g carrier, such as 10, 15, 20, 25, 30, 35, 40 mg peptide/g carrier, particularly preferably 20 mg Peptide/g carrier.
  • the method step (1) of the third aspect can be carried out at any suitable temperature and pH.
  • the temperature may be about 20-37°C, such as about 20-35°C, 25-37°C, 25-35°C, 20-30°C, 25-35°C, especially about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37°C.
  • the pH value can be any suitable pH, for example about 4.0-10.0, such as about 5.0-9.0, 6.0-8.0 or 6.0-7.0, especially about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0.
  • the target protein linked to the SpyTag peptide in step (2) of the third aspect of the method can be a separated and purified protein, such as a fusion protein separated and purified by techniques known in the art; or it can contain the peptide and the target.
  • the fusion protein can be obtained by recombinant expression in prokaryotes, yeast or higher eukaryotic cells.
  • prokaryotes include Escherichia, Bacillus, Salmonella, and Pseudomonas Bacteria of the genera Pseudomonas and Streptomyces.
  • the recombinant cell is an Escherichia cell, preferably Escherichia coli.
  • the recombinant cells used are E. coli BL21 (DE3) strain cells (Novagen).
  • the target protein of the present invention can be any protein, such as red fluorescent protein (RFP) and glutaryl-7-aminocephalosporanic acid acylase (GA) listed in the examples of the present invention.
  • the cell lysate can be obtained by methods known in the art, for example, methods for disrupting cells include, but are not limited to: ultrasound, homogenization, high pressure (for example, in a French press), hypotonic (osmolysis), detergent , Lyase, organic solvent or a combination thereof.
  • the fragmentation is performed under the first pH condition (ie, weakly alkaline pH, such as pH 7.2-8.5, preferably pH 7.4), so that the cell membrane of the host cell is fragmented, and the protein supernatant is removed from the fragmentation. Released from the bacteria, and still maintain a soluble state.
  • the purified fusion protein can be obtained by affinity tag purification.
  • the affinity tag is a common 6 ⁇ His tag, which is located at the C-terminus of the target protein.
  • the released protein supernatant is purified by protein affinity chromatography. After the cell membrane of the recombinant cell is broken, the supernatant can be collected by centrifugation to remove the insoluble precipitate, and then purified by affinity chromatography through the His tag of the fusion protein.
  • the modified carrier and the fusion protein are contacted under conditions that allow the SpyCatcher peptide and the SpyTag peptide to form isopeptide bonds.
  • the conditions that allow SpyCatcher peptides and SpyTag peptides to form isopeptide bonds (Spy reactions occur) are known in the art (as described above).
  • contact is to physically associate the modified carrier with the protein, such as mixing two substances in a solution, or by adding another substance to a solution containing one substance (for example, a fusion protein) (such as modified vectors).
  • the method step (3) of the third aspect includes contacting the SpyCatcher modified carrier with the fusion protein, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48 hours.
  • the contact is centrifuged at about 20-50 rpm, such as about 25-40, 25-35, 25-30 rpm.
  • the method step (3) of the third aspect can be carried out at any suitable temperature and pH.
  • the temperature can be about 4-37°C, such as about 5-35°C, 10-35°C, 15-35°C, 20-35°C, 25-37°C, 25-35°C, 20-30°C, 25°C -35°C, especially about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37°C.
  • the pH value can be any suitable pH, for example about 4.0-10.0, such as about 5.0-9.0, 6.0-8.0 or 6.0-7.0, especially about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0.
  • pH 6.0-7.0 is particularly preferred.
  • step (3) of the method of the third aspect includes incubating the fusion protein of the SpyTag peptide and the target protein with the carrier connected to the SpyCatcher peptide obtained in step (1); and optionally washing with a washing buffer Remove unimmobilized fusion protein.
  • the washing buffer is a protein washing buffer commonly used in the art, such as phosphate buffered saline (PBS), Tris-HCl buffer, and the like.
  • PBS phosphate buffered saline
  • Tris-HCl buffer Tris-HCl buffer
  • the carrier connected to the fusion protein can be separated by any suitable technique known in the art, such as centrifugation.
  • a protein immobilization method based on Spy reaction comprising:
  • a kit which includes a carrier, a SpyCatcher peptide, and optionally a fusion protein of the SpyTag peptide and the target protein.
  • the carrier, SpyCatcher peptide, SpyTag peptide, protein, etc. are as described above.
  • the carrier and SpyCatcher peptide exist separately. Before use, the carrier and the peptide can be contacted (for example, mixed in a solution) to prepare the SpyCatcher peptide-modified carrier described in the first aspect.
  • the carrier and the SpyCatcher peptide are in the form of the SpyCatcher peptide-modified carrier described in the first aspect.
  • step refers to the presence or absence of the step.
  • the term “about” refers to a range of values that includes a specific value, and those skilled in the art can reasonably consider it to be similar to the specific value. In certain embodiments, the term “about” refers to within the standard error of using a measurement commonly accepted in the art. For example, in certain embodiments, about refers to +/- 10% or 5% of the specified value.
  • the method provided by the present disclosure is compatible with existing commercial immobilized carriers, does not require new carrier surface chemistry, does not require additional enzyme catalysts such as sortase, and can directly use cell lysate containing the desired protein for immobilization.
  • the immobilization process of the disclosed method in the case of an epoxy carrier only requires one step, the ligation reaction is specific, the efficiency is high, the conditions are mild (room temperature, neutral pH), and no additional chemical reagents are required.
  • the immobilized carrier improved by the disclosed method has good versatility, high immobilization efficiency and vitality recovery rate, good uniformity, and can reduce the work of screening the immobilized carrier.
  • oligonucleotide primers used in the present invention are shown in Table 1.
  • the underlined parts of a primer are the recognition sites of restriction enzymes Nde I, Hind III, Sac I and Xho I.
  • the pET30a(+)-PT linker-SpyCatcher-His expression plasmid (SEQ ID NO: 17) constructed in the embodiment of the present invention is used to express the Ptlingker-SpyCatcher-His protein.
  • the plasmid map is shown in Figure 2a.
  • PCR reaction procedure a) 98°C for 30sec; b) 98°C for 10sec; c) 57°C for 15sec; d) 72°C for 15sec; e) Steps b-d, 30 cycles; f) 72°C for 2min; g) 4°C hold.
  • the PCR amplified products were detected by 1% agarose gel electrophoresis, and the PCR amplified the target DNA bands that were consistent with expectations. Purify and recover the target DNA using an ultra-thin DNA product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP203-02), and store it at -20°C for later use.
  • an ultra-thin DNA product purification kit Tiangen Biochemical Technology (Beijing) Co., Ltd., DP203-02
  • the purified and recovered target DNA was double digested with restriction enzymes Nde I (NEB, RO111L) and Xho I (NEB, R0146L), and then it was digested with the plasmid pET-30a(+) (Novagen )
  • the ligation product was transformed into E. coli BL21 (DE3) (Novagen) competent cells, and the transformed cells were spread on LB (Luria-Bertani medium) supplemented with 50 ⁇ g/mL Kanamycin (Kan) ) On the plate, select positive clones, extract plasmids, and sequence them.
  • the sequencing results show that the sequence of the cloned pET30a(+)-PT linker-SpyCatcher-His is correct.
  • the pET30a(+)-SpyTag-GS linker-RFP-His (SEQ ID NO: 18) expression plasmid constructed in the embodiment of the present invention is used to express the SpyTag-RFP (SEQ ID) fusion protein of SpyTag and red fluorescent protein (RFP) NO: 24), the plasmid map is shown in Figure 2b;
  • pET30a(+)-SpyTag-GS linker-GA-His expression plasmid is used to express SpyTag and glutaryl-7-aminocephalosporanic acid acylase (glutaryl-
  • the fusion protein SpyTag-GA (SEQ ID NO: 25) of 7-aminocephalosporanic acid acylase, GA, and its plasmid map is shown in Figure 2c.
  • PCR reaction procedures a) 98°C for 30sec; b) 98°C for 10sec; c) 57°C for 20sec; d) 72°C for 30sec; e) Steps b-d, 30 cycles; f) 72°C for 2min; g) 4°C hold.
  • PCR amplified products were detected by 1% agarose gel electrophoresis. As a result, PCR amplified a target DNA band that was consistent with expectations. Purify and recover the target DNA using an ultra-thin DNA product purification kit, and store it at -20°C for later use.
  • the purified and recovered target DNA was double digested with restriction enzymes Nde I and Hind III (NEB, R3104L), and then ligated with the plasmid pET-30a(+) double digested with the same enzymes to transform the ligation product To Ecoli.BL21(DE3) competent cells, spread the transformed cells on an LB plate supplemented with 50 ⁇ g/mL Kan, select positive clones, extract plasmids, and sequence them.
  • the sequencing results show that the cloned pET30a(+) -SpyTag-Gslinker-RFP-His sequence is correct.
  • PCR reaction procedure a) 98°C for 30sec; b) 98°C for 10sec; c) 57°C for 20sec; d) 72°C for 90sec; e) Steps b-d, 30 cycles; f) 72°C for 4min; g) 4°C hold.
  • PCR amplified products were detected by 1% agarose gel electrophoresis. As a result, PCR amplified a target DNA band that was consistent with expectations. Purify and recover the target DNA using an ultra-thin DNA product purification kit, and store it at -20°C for later use.
  • the purified and recovered target DNA was double digested with restriction enzymes Sac I (NEB, R0156L) and Hind III, and then digested with the plasmid pET30a(+)-SpyTag-Gslinker-GA-His double digested with the same enzymes Ligation, transform the ligation product into E. coli BL21(DE3) competent cells, spread the transformed cells on an LB plate supplemented with 50 ⁇ g/mL Kan, select positive clones, extract plasmids, and sequence them.
  • the sequencing results show that The cloned pET30a(+)-SpyTag-Gslinker-GA-His sequence is correct.
  • Example 2 Expression, purification and activity determination of fusion protein
  • IPTG isopropyl- ⁇ -D- Isopropyl ⁇ -D-1-thiogalactopyranoside
  • Example 1 The strain constructed in Example 1 (containing the plasmid pET30a(+)-SpyTag-GS linker-GA-His) was inoculated into LB liquid medium containing 50 ⁇ g/mL Kan, and the inoculum was 1:50 overnight.
  • the cells obtained in step 2.1 were harvested and resuspended to 50 OD/mL with PBS buffer (0.1M, pH 7.0). Cells were disrupted by sonication on ice (disruption conditions: power 200W, sonication time 3sec, interval time 3sec, and sonication 99 times). After the sonication is completed, the supernatant and precipitate of the buffer are separated by centrifugation. In order to remove the mixed soluble components in the precipitate as much as possible, wash the obtained precipitate twice with an equal volume of buffer. The supernatant and pellet resuspension were directly used for SDS-PAGE determination.
  • Example 2.1 The cells in Example 2.1 were harvested and resuspended to 50 OD/mL with PBS buffer (0.1M, pH 7.0). The cells were disrupted with a high-pressure homogenizer (PhD Technology International LLC, USA) (disruption conditions: 12000 psi pressure, 3 cycles of disruption). After the high-pressure homogenate is broken, the supernatant and precipitate of the lysate are separated by centrifugation. The supernatant was collected and purified by affinity chromatography on a nickel column. The samples before and after purification and the flow-through fluid were collected, and the purification effect was detected by SDS-PAGE.
  • PBS buffer 0.1M, pH 7.0
  • the cells were disrupted with a high-pressure homogenizer (PhD Technology International LLC, USA) (disruption conditions: 12000 psi pressure, 3 cycles of disruption). After the high-pressure homogenate is broken, the supernatant and precipitate of the lysate are separated
  • the purified protein supernatant was dialyzed, and the dialysis buffer was PBS buffer (0.1M, pH 7.0).
  • Lanes 6, 7, and 8 are the fusion proteins SpyTag-GSlinker-GA (for simplicity, hereinafter referred to as SpyTag-GA), PT linker-SpyCatcher (for simplicity, hereinafter referred to as SpyCatcher) and SpyTag-Gslinker-RFP (for simplicity, Hereinafter referred to as SpyTag-RFP), the purity of the three fusion proteins after purification were 95%, 81%, and 88%, respectively, which can be used for subsequent immobilization experiments.
  • SpyTag-GA fusion proteins
  • PT linker-SpyCatcher for simplicity, hereinafter referred to as SpyCatcher
  • SpyTag-Gslinker-RFP for simplicity, Hereinafter referred to as SpyTag-RFP
  • GA catalyzes the hydrolysis of glutaryl-7-aminocephalosporanic acid (GL-7-ACA) to 7-aminocephalosporanic acid (7-ACA).
  • the primary amino group of 7-ACA reacts with p-dimethylaminobenzaldehyde (pDAB) to form a yellow Schiff base with a maximum absorbance at 415nm.
  • pDAB p-dimethylaminobenzaldehyde
  • Free GA enzyme activity determination 20 ⁇ L enzyme solution (in 0.1M PBS buffer at pH 7.0) and 20 ⁇ L substrate (1% (w/v) in 0.1M PBS buffer at pH 7.0) GL-7-ACA ) Mix and incubate at 37°C for 10 minutes. Add 140 ⁇ L of 20% (v/v) acetic acid and 0.05M NaOH mixture (2:1, v/v) to stop the reaction, and then add 20 ⁇ L of 0.5% (w/v) pDAB. The mixture was incubated for another 10 minutes at 25°C, and then the absorbance at 415 nm was measured.
  • One GA activity unit (U) is defined as the amount of enzyme required to produce 1 ⁇ mol 7-ACA per minute at 37°C and pH 7.0.
  • ⁇ A/min represents the change in absorbance per minute, which is the slope
  • Vt represents the total volume of the reaction solution (0.2mL).
  • Vs represents the volume of the sample enzyme solution (0.02mL);
  • Free RFP fluorescence detection After the purified RFP is properly diluted, add 200 ⁇ L to a black flat-bottom 96-well plate (Corning, 3925), and use a grating-type multifunctional microplate reader (infinite M200PRO, TECAN) to detect RFP fluorescence at 37°C Intensity, its excitation wavelength is 588nm, emission wavelength is 635nm, gain value is 100% [Anuar I N A K, Banerjee A, Keeble A H, et al. Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox [J].Nature communications,2019,10(1):1734.].
  • the present invention first obtains the SpyCatcher modified carrier, and then uses Spy chemistry to immobilize the SpyTag fusion protein in one step.
  • SpyCatcher-LX-1000EP a SpyCatcher modified vector
  • SpyCatcher fixed amount (mg) total protein amount of supernatant before fixation (mg)-total protein amount of supernatant after fixation (mg)
  • the obtained immobilized SpyCatcher-LX-1000EP was added to 12-fold volume of blocking buffer (3M glycine, pH 8.5), and reacted with a miniature rotary mixer (Gilson, Roto-Mini Plus) at 25° C. and 25 rpm for 24 hours. After the blocking is completed, centrifuge to remove the blocking solution, wash 3 times with PBS buffer (0.1M, pH 7.0), and store at 4°C.
  • blocking buffer 3M glycine, pH 8.5
  • SpyCatcher-LX-1000EP was used to immobilize purified SpyTag-RFP at different pH.
  • the method of the present invention Weigh 50mg SpyCatcher-LX-1000EP, wash with 0.1M PBS buffer (pH 5.0, 6.0, 7.0, 8.0, respectively) for 3 times, add 0.75mg purified SpyTag-RFP (3.75mg/mL) (pH is 5.0, 6.0, 7.0, 8.0, respectively), and react with a miniature rotary mixer (Gilson, Roto-Mini Plus) at 25°C and 25 rpm for 2 hours.
  • a miniature rotary mixer Galves, Roto-Mini Plus
  • Blocking control Weigh 50mg of the epoxy carrier after the blocking reaction, wash it 3 times with 0.1M PBS buffer (pH 5.0, 6.0, 7.0, 8.0), and add 0.75mg of purified SpyTag-RFP (3.75mg/ mL) (pH 5.0, 6.0, 7.0, 8.0, respectively), and react with a miniature rotary mixer (Gilson, Roto-Mini Plus) at 25°C and 25 rpm for 2 hours. As a closed control group.
  • Epoxy method Weigh 50mg LX-1000EP, wash 3 times with 0.1M PBS buffer (pH 5.0, 6.0, 7.0, 8.0), add 0.75mg purified SpyTag-RFP (3.75mg/mL) (pH Respectively 5.0, 6.0, 7.0, 8.0), the reaction was performed with a miniature rotary mixer (Gilson, Roto-Mini Plus) at 25°C and 25 rpm for 2 hours. As a control group.
  • the immobilized RFP was separated by centrifugation. Wash 5 times with PBS buffer (0.1M, pH 7.0) to obtain immobilized RFP. To calculate the fixed amount and efficiency of immobilized RFP, the relevant calculation formula is as follows:
  • RFP fixed amount (mg) total protein of supernatant before fixation (mg)-total protein of supernatant after fixation (mg)-total protein of washing liquid supernatant (mg)
  • the immobilization results are shown in Table 6.
  • Immobilization of protein in the range of pH 5-8 The efficiency of immobilization and activity recovery rate of epoxy method increases with the increase of pH, the immobilization efficiency is 31-41%, and the activity recovery rate is 22-43%;
  • the method of the invention has the highest immobilization efficiency and vitality recovery rate when the pH is 6-7, the immobilization efficiency is 51-62%, and the vitality recovery rate is 51-67%.
  • SpyCatcher-LX-1000EP was used to immobilize purified SpyTag-RFP at different temperatures.
  • the method of the present invention Weigh 50 mg of SpyCatcher-LX-1000EP, wash with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.75 mg of purified SpyTag-RFP (3.75 mg/mL), and use a micro rotating mixer ( Gilson, Roto-Mini Plus) were reacted at 4°C, 25°C, 37°C and 25rpm for 2h.
  • Blocking control Weigh 50mg of epoxy carrier after blocking reaction, wash with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.75mg of purified SpyTag-RFP (3.75mg/mL), and mix with micro-rotation The instrument (Gilson, Roto-Mini Plus) was reacted at 4°C, 25°C, 37°C and 25rpm for 2h. As a closed control group.
  • Epoxy method Weigh 50mg LX-1000EP, wash 3 times with PBS buffer (0.1M, pH 7.0), add 0.75mg purified SpyTag-RFP (3.75mg/mL), use a miniature rotary mixer (Gilson, Roto-Mini Plus) was reacted at 4°C, 25°C, 37°C and 25rpm for 2h. As a control group.
  • the immobilized RFP was separated by centrifugation. Wash 5 times with PBS buffer (0.1M, pH 7.0) to obtain immobilized RFP. To calculate the fixed amount and efficiency of immobilized RFP, the relevant calculation formula is as follows:
  • RFP fixed amount (mg) total protein amount of supernatant before fixation (mg)-total protein amount of supernatant after fixation (mg)-total protein amount of washing liquid supernatant (mg)
  • the immobilization results are shown in Table 7. Immobilization of protein at temperatures of 4°C, 25°C and 37°C: As the temperature rises, the epoxy method increases the immobilization efficiency and vitality recovery rate, the immobilization efficiency is 26-40%, and the vitality recovery rate is 34 -47%; the method of the present invention is not sensitive to temperature, the immobilization efficiency is 62% at 4°C, 25°C and 37°C, and the activity recovery rate is 65-67%.
  • the method of the present invention Weigh 50 mg of SpyCatcher-LX-1000EP, wash with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.75 mg of purified SpyTag-RFP (3.75 mg/mL), and use a micro rotating mixer ( Gilson, Roto-Mini Plus) reacted at 25°C and 25rpm for 2h.
  • Epoxy method Weigh 50mg LX-1000EP, wash 3 times with PBS buffer (0.1M, pH 7.0), add 0.75mg purified SpyTag-RFP (3.75mg/mL), use a miniature rotary mixer (Gilson, Roto-Mini Plus) react for 2h at 25°C and 25rpm. As a control group.
  • Blocking control Weigh 50mg of epoxy carrier after blocking reaction, wash with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.75mg of purified SpyTag-RFP (3.75mg/mL), and mix with micro-rotation The instrument (Gilson, Roto-Mini Plus) reacted at 25°C and 25 rpm for 2 hours. As a closed control group.
  • the immobilized RFP was separated by centrifugation. Wash 5 times with PBS buffer (0.1M, pH 7.0) to obtain immobilized RFP. To calculate the fixed amount and efficiency of immobilized RFP, the relevant calculation formula is as follows:
  • RFP fixed amount (mg) total protein amount of supernatant before fixation (mg)-total protein amount of supernatant after fixation (mg)-total protein amount of washing liquid supernatant (mg)
  • the analysis result of SDS-PAGE is shown in Figure 5a.
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software, and the immobilization efficiency of the fusion protein SpyTag-RFP can be calculated.
  • the results are shown in Table 8.
  • the immobilization efficiency of the closed control immobilization SpyTag-RFP was 13.4%
  • the immobilization efficiency of the epoxy immobilization method was 29.6%
  • the immobilization efficiency of the method of the present invention was 66.0%.
  • the method of the present invention Weigh 50mg SpyCatcher-LX-1000EP, wash 3 times with PBS buffer (0.1M, pH 7.0), add 0.2mL of E. coli cell lysate supernatant overexpressing SpyTag-RFP, and mix with micro-rotation
  • the instrument (Gilson, Roto-Mini Plus) reacted at 25°C and 25 rpm for 2 hours.
  • Epoxy method Weigh 50 mg of LX-1000EP, wash with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.2 mL of E. coli cell lysate supernatant overexpressing SpyTag-RFP, and use a micro-rotating mixer ( Gilson, Roto-Mini Plus) reacted at 25°C and 25rpm for 2h. As a control group.
  • Blocking control Weigh 50mg of the epoxy carrier after the blocking reaction, wash it with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.2mL of the supernatant of E. coli cell lysate overexpressing SpyTag-RFP, and spin it with a micro spin The mixer (Gilson, Roto-Mini Plus) reacted at 25°C and 25 rpm for 2 hours. As a closed control group.
  • the immobilized RFP was separated by centrifugation. Wash 5 times with PBS buffer (0.1M, pH 7.0). Obtain the immobilized SpyTag-RFP. Calculate the fixed amount, immobilization efficiency and fluorescence recovery rate of immobilized RFP, the relevant calculation formula is as follows:
  • RFP fixed amount (mg) total protein amount of supernatant before fixation (mg)-total protein amount of supernatant after fixation (mg)-total protein amount of washing liquid supernatant (mg)
  • the input amount of fusion protein is estimated by SDS-PAGE.
  • the method of the present invention Weigh 50mg of SpyCatcher-LX-1000EP, wash with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.75mg of purified SpyTag-GA (3.75mg/mL), and use a micro rotating mixer (Gilson, Roto-Mini Plus) React at 25°C and 25rpm for 2h.
  • Epoxy method Weigh 50mg LX-1000EP, wash 3 times with PBS buffer (0.1M, pH 7.0), add 0.75mg purified SpyTag-GA (3.75mg/mL), use a micro-rotating mixer (Gilson , Roto-Mini Plus) react at 25°C and 25rpm for 2h. As a control group.
  • Blocking control Weigh 50 mg of the epoxy carrier after the blocking reaction, wash it with PBS buffer (0.1M, pH 7.0) for 3 times, add 0.75 mg of purified SpyTag-GA (3.75 mg/mL), and mix it with a micro spinner. The homogenizer (Gilson, Roto-Mini Plus) reacted at 25°C and 25 rpm for 2 hours. As a closed control group.
  • the immobilized enzyme GA is separated by centrifugation. Wash 5 times with PBS buffer (0.1M, pH 7.0). Obtain immobilized GA. Calculate the fixed amount, immobilization efficiency and enzyme activity recovery rate of immobilized enzyme GA, the relevant calculation formula is as follows:
  • GA fixed amount (mg) total protein amount of supernatant before fixation (mg)-total protein amount of supernatant after fixation (mg)-total protein amount of washing liquid supernatant (mg)
  • the analysis result of SDS-PAGE is shown in Figure 7.
  • the optical density analysis of the target band was performed using ImageJ gel quantitative analysis software, and the immobilization efficiency of the fusion protein SpyTag-GA can be calculated.
  • the results are shown in Table 10.
  • the efficiency of immobilization of SpyTag-GA in the closed control is 28.4%
  • the immobilization efficiency of the epoxy immobilization method is 29.4%
  • the immobilization efficiency of the method of the present invention is increased to 91.5%.
  • the immobilized enzyme GA is separated by centrifugation. Wash 5 times with PBS buffer (0.1M, pH 7.0). Obtain immobilized SpyTag-GA. To calculate the fixed amount, immobilization efficiency and vitality recovery rate of SpyTag-GA, the relevant calculation formula is as follows:
  • GA fixed amount (mg) total protein amount of supernatant before fixation (mg)-total protein amount of supernatant after fixation (mg)-total protein amount of washing liquid supernatant (mg)
  • the immobilization efficiency of the fusion protein SpyTag-GA in the cell lysate can be calculated.
  • the results are shown in Table 11.
  • the efficiency of immobilization of SpyTag-GA in the closed control is 28.5%
  • the immobilization efficiency of the epoxy method is 25.0%
  • the immobilization efficiency of the method of the present invention is increased to 86.2%.
  • the input amount of fusion protein is estimated by SDS-PAGE.
  • Fluorescence detection of immobilized SpyTag-RFP Weigh 2 mg (appropriate amount) of immobilized RFP, add it to a black flat-bottomed 96-well plate, and add 0.2mL PBS buffer (0.1M, pH 7.0) to fully resuspend it.
  • PBS buffer 0.1M, pH 7.0
  • the functional microplate reader detects the fluorescence intensity of the immobilized SpyTag-RFP at 37°C.
  • the excitation wavelength is 588nm
  • the emission wavelength is 635nm
  • the gain value is 100%.
  • Enzyme activity determination of immobilized SpyTag-GA weigh 4mg of immobilized SpyTag-GA, add 0.5mL 0.1M PBS buffer (pH 7.0), and 0.5mL substrate (1 in 0.1M PBS buffer pH 7.0) %(W/v)GL-7-ACA) mixed and incubated at 37°C for 10 minutes. Then take out 0.2mL reaction solution, first add 0.7mL 20% (v/v) acetic acid and 0.05M NaOH mixture (2:1, v/v), and then add 0.1mL 0.5% (w/v) pDAB. The mixture was incubated for another 10 minutes at 25°C, and then the absorbance at 415 nm was measured.
  • ⁇ A/min represents the change in absorbance per minute, which is the slope
  • Vt represents the total volume of the reaction solution (1mL);
  • the results of the immobilized SpyTag-RFP/SpyTag-GA activity test are shown in Table 12.
  • the activity recovery rates of SpyTag-RFP and SpyTag-GA were fixed in the blocking control and the activity recovery rates were 2.8% and 1.1%, respectively.
  • the activity recovery rates of the epoxy method were 19.6% and 2.7%, respectively, while the activity recovery rate of the method of the present invention was increased to 49.4%.
  • the immobilization method can also directly immobilize SpyTag-RFP/SpyTag-GA from the cell lysate.
  • the activity recovery rate of the epoxy method is 36.3% and 3.4%, respectively, and the activity recovery rate of the method of the present invention is increased to 85.0% and 91.2%.
  • the method of the present invention and the epoxy method select 6 kinds of epoxy carriers and 2 kinds of proteins (including the purified protein SpyTag-RFP and the cell lysate containing the target protein SpyTag-RFP and SpyTag-GA) for immobilization, and the immobilization
  • Table 17 shows the statistical data of the immobilization efficiency of the method of the present invention and the epoxy method.
  • the method of the present invention adopts SpyCatcher modified modified resin, the average and median value of immobilization efficiency are higher than epoxy method, and the coefficient of variation is much lower than epoxy method, indicating that the immobilization efficiency of the method of the present invention is higher than that of epoxy method. Higher and better uniformity.
  • the immobilization efficiency of the SpyCatcher modified resin and the epoxy carrier immobilized protein in the method of the present invention Comparing the immobilization efficiency of the SpyCatcher modified resin and the epoxy carrier immobilized protein in the method of the present invention, the result is shown in FIG. 10. For most epoxy carriers, the immobilization efficiency of the method of the present invention is significantly higher or not lower than that of the epoxy method.
  • the method of the present invention and the epoxy method select 6 kinds of epoxy carriers and 2 kinds of proteins (including the purified protein SpyTag-RFP and the cell lysate containing the target protein SpyTag-RFP and SpyTag-GA) for immobilization, and their vitality is recovered
  • the comparison of rates is summarized in Figure 11.
  • the statistical data of the immobilization efficiency of the method of the present invention and the epoxy method are shown in Table 18.
  • the method of the present invention adopts the modified resin modified by SpyCatcher, and the average and median value of vitality recovery rate are higher than the epoxy method, while the range, interquartile range and coefficient of variation are much lower than the epoxy method, indicating the method of the present invention
  • the immobilization efficiency is higher and the uniformity is better than the epoxy method.
  • the prior art method has an unstable effect on protein immobilization, and is more sensitive to protein types and epoxy carrier types, and a large amount of epoxy carrier screening work is required for a certain target protein.
  • the method of the present invention improves the epoxy carrier, improves the uniformity of the carrier, and reduces the screening work for the carrier.
  • the relative activity comparison of the immobilized protein by the method of the present invention and the epoxy method is summarized as shown in FIG. 13.
  • the statistical data of the immobilization efficiency of the method of the present invention and the epoxy method are shown in Table 19.
  • the method of the present invention adopts SpyCatcher modified modified carrier to immobilize the protein, and its relative activity average and median value are 89.1% and 96.9%, respectively.
  • There is almost no loss of activity after protein immobilization while the protein immobilized by epoxy method has The average and median values were 57.3% and 48.4%, respectively, and the loss of vitality was nearly half.
  • the relative activity variation coefficient of the immobilization of the method of the present invention is far lower than that of the epoxy method, indicating that the immobilization efficiency of the method of the present invention is higher than that of the epoxy method and the uniformity is better.
  • the prior art methods have unstable effects on protein immobilization, and are more sensitive to protein types and epoxy carrier models, and a large amount of epoxy carrier screening work is required for a certain target protein.
  • the method of the present invention improves the epoxy carrier, improves the uniformity of the carrier, and reduces the screening work for the carrier.
  • Example 6 Comparison of the immobilization effect of different batches of epoxy carriers using the method of the present invention and the existing method
  • Example 7 SpyCatcher003 modified carrier fixes SpyTag003-GA in cell lysate
  • oligonucleotides and primers used in this example are shown in Table 20.
  • the underlined parts of a primer are the recognition sites of restriction enzymes Xba I and Sac I, respectively.
  • the pET30a(+)-PTlinker-SpyCatcher003-His expression plasmid (SpyCatcher003, SEQ ID NO: 23) constructed in this example was used to express the PTlinker-SpyCatcher003-His protein, and the construction method was the same as that in Example 1.1.
  • oligonucleotide sequences SpyTag003-GS-1, 2, 3 and 4 in Table 20 Use the oligonucleotide sequences SpyTag003-GS-1, 2, 3 and 4 in Table 20 to perform PCR splicing and amplification to obtain the Xba I-SpyTag003-Gslinker-Sac I polynucleotide fragment.
  • the PCR splicing system is shown in Table 21 Shown.
  • the PCR reaction uses Q5 high-fidelity DNA polymerase, and the PCR amplification system and procedures are shown in Table 22.
  • PCR splicing reaction procedure a) 98°C 30sec; b) 98°C 10sec; c) 62°C 20sec; d) 72°C 10sec; e) step b)-d), 10 cycles; f) 72°C 2min; g) 10 °C hold.
  • PCR amplification reaction procedure a) 98°C for 30sec; b) 98°C for 10sec; c) 66°C for 20sec; d) 72°C for 10sec; e) steps b-d, 30 cycles; f) 72°C for 2min; g) 4°C hold.
  • the PCR amplified products were detected by 2% agarose gel electrophoresis, and the results showed that the PCR amplified target DNA bands consistent with expectations. Purify and recover the target DNA using an ultra-thin DNA product purification kit, and store it at -20°C for later use.
  • the purified and recovered target DNA was double digested with restriction enzymes Xba I and Sac I (NEB, R0156L), and then digested with the plasmid pET30a(+)-SpyTag-Gslinker-GA-His double-cut with the same enzymes Ligation, transform the ligation product into E. coli BL21(DE3) competent cells, spread the transformed cells on LB plates supplemented with 50 ⁇ g/mL kanamycin (Kan), select positive clones, extract plasmids, and perform Sequencing results showed that the sequence of the cloned pET30a(+)-SpyTag003-Gslinker-GA-His was correct.
  • Kan Kanamycin
  • the preparation method and the calculation of immobilization efficiency are the same as in Example 3.1, and the results are shown in Table 23.
  • the immobilization efficiency was 90.1%.
  • the obtained immobilized SpyCatcher003-LX-1000EP was added to a 12-fold volume of blocking buffer (3M glycine, pH 8.5), and reacted with a miniature rotary mixer (Gilson, Roto-Mini Plus) at 25°C and 25 rpm for 24 hours. After the blocking is completed, centrifuge to remove the blocking solution, wash 3 times with PBS buffer (0.1M, pH 7.0), and store at 4°C.
  • blocking buffer 3M glycine, pH 8.5
  • the fixation method and the calculation of the fixation efficiency are the same as in Example 4.3.4, and the results are shown in Table 24.
  • the efficiency of immobilization of SpyTag003-GA in closed control is 14.9%
  • the immobilization efficiency of epoxy method is 31.9%
  • the immobilization efficiency of the method of the present invention is 26.1%.
  • the input amount of fusion protein is estimated by SDS-PAGE.
  • the activity detection method and enzyme activity calculation are the same as in Example 4.4, and the results are shown in Table 25.
  • the activity recovery rate of the epoxy method is 33.1%, and the activity recovery rate of the method of the present invention is 23.8%.
  • the present invention provides an enzyme immobilization method based on the SpyCatcher/SpyTag reaction.

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Abstract

一种SpyCatcher肽修饰的载体，固定了蛋白质的载体以及蛋白固定化方法。所述方法包括将SpyCatcher肽与载体连接以获得SpyCatcher肽修饰的载体，然后基于SpyCatcher-SpyTag反应，将含有SpyTag的目的蛋白固定在SpyCatcher修饰的载体上。使用该方法的蛋白固定化过程快速、条件温和、不需要额外的化学试剂，固定化效率和活性回收率高，均一性好。

Description

蛋白固定化用载体及其制备方法 技术领域
本发明涉及生物工程领域，特别是蛋白固定化例如酶固定化领域，具体涉及一种蛋白固定化方法、用于固定蛋白的修饰的载体以及固定了蛋白的修饰的载体。
背景技术
与游离蛋白质相比，固定化蛋白质(如酶)可重复使用、易与反应体系分离、稳定性得到提高，因此在涉及蛋白质生物结合或催化反应的应用中，固定化蛋白质比游离蛋白质具有明显的优势。
聚合环氧载体(如聚丙烯、聚苯乙烯)由于其机械稳定性较高，在工业酶的固定化中具有广泛的应用[Boller,Thomas,Christian Meier,and Stefan Menzler."Eupergit oxirane acrylic beads:how to make enzymes fit for biocatalysis."Organic Process Research & Development 6.4(2002):509-519；Mateo,Cesar,et al."Immobilization of enzymes on heterofunctional epoxy supports."Nature Protocols 2.5(2007):1022；McAuliffe,Joseph C."Industrial enzymes and biocatalysis."Handbook of industrial chemistry and biotechnology.Springer,Boston,MA,2012.1183-1227]。但是，环氧载体固定化蛋白质的活力回收率往往很低，Boller T等统计了采用环氧载体固定化23种工业酶的情况，其固定化活力回收率大部分在30％-40％或更低[Boller,Thomas,Christian Meier,and Stefan Menzler."Eupergit oxirane acrylic beads:how to make enzymes fit for biocatalysis."Organic Process Research & Development 6.4(2002):509-519]。
环氧活化载体(以下简称环氧载体)可以通过载体上的环氧基团与蛋白表面的多种亲核基团(例如赖氨酸、半胱氨酸、组氨酸、酪氨酸等)，因此可以实现蛋白的多点共价固定化。环氧载体的种类和性质对蛋白固定化的结果影响很大，对同一种酶，使用不同的环氧载体，其固定化效率和活力回收率往往差别很大[Seip,John E.,et al."Glyoxylic acid production using immobilized glycolate oxidase and catalase."Bioorganic & medicinal chemistry 2.6(1994):371-378]。环氧载体的物理性能使其非常适合用于工业催化，研究者采用多种办法改良环氧载体，其中最常见的改良策略是在环氧载体上增加其它功能基团(如氨基、高活性二硫键、羧基、金属螯合基团等)。[Mateo,Cesar,et al."Immobilization of enzymes on heterofunctional epoxy supports."Nature Protocols 2.5(2007):1022]。而且，在进行环氧载体上环氧基团修饰时，还需要优化修饰的环氧基团比例。[Mateo,Cesar,et al."Multifunctional epoxy supports:a new tool to improve the covalent immobilization of proteins.The promotion of physical adsorptions of proteins on the supports before their covalent linkage."Biomacromolecules 1.4(2000):739-745]。这些环氧载体的修饰方法虽然对某些特定蛋白会有明显效果，但对于不同的蛋白，需对环氧载体做不同的修饰，而且修饰的过程需要进行优化，其效果难以预料，修饰的环氧载体仍然需要较多的试错和筛选工作。综上，现有基于环氧载体的蛋白固定化技术对蛋白和环氧载体种类敏感，为得 到较高的固定化蛋白活力回收率，需要进行大量环氧载体修饰的优化或环氧载体筛选。
定向固定化的蛋白具有均一的取向，能最大限度地保持蛋白的活性，因此蛋白的定向固定化在工业生物催化、临床诊断和蛋白相互作用分析等领域有着广泛的应用[Wong,Lu Shin,Farid Khan,and Jason Micklefield."Selective covalent protein immobilization:strategies and applications."Chemical reviews 109.9(2009):4025-4053.；Wong,Lu Shin,Farid Khan,and Jason Micklefield."Selective covalent protein immobilization:strategies and applications."Chemical reviews 109.9(2009):4025-4053.；Meldal,Morten,and Sanne Schoffelen."Recent advances in covalent,site-specific protein immobilization."F1000Research 5(2016).；Liese,Andreas,and Lutz Hilterhaus."Evaluation of immobilized enzymes for industrial applications."Chemical Society Reviews 42.15(2013):6236-6249]。由于在大多数蛋白中存在多个相同的氨基酸，传统的靶向特定氨基酸(如赖氨酸、半胱氨酸)的固定化方法难以实现蛋白的定向固定化[Mateo,Cesar,et al."Immobilization of enzymes on heterofunctional epoxy supports."Nature Protocols 2.5(2007):1022]。例如，在载体上修饰寡聚甘氨酸，再用分选酶A(Sortase A)催化蛋白C端的LPXTG基序，可以将蛋白连接在寡聚甘氨酸的N端而实现定向蛋白固定化；甲酰基甘氨酸生成酶(FGE)修饰CXPXR上的半胱氨酸残基(C)后，可通过希夫碱反应连接在载体的氨基上实现蛋白的定向固定化[Popp,Maximilian W.,et al."Sortagging:a versatile method for protein labeling."Nature chemical biology 3.11(2007):707-708；Raeeszadeh-Sarmazdeh,Maryam,Ranganath Parthasarathy,and Eric T.Boder."Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases."Colloids and Surfaces B:Biointerfaces 128(2015):457-463；Rabuka,David,et al."Site-specific chemical protein conjugation using genetically encoded aldehyde tags."Nature protocols 7.6(2012):1052；Cho,Hwayoung,and Justyn Jaworski."Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins."Colloids and Surfaces B:Biointerfaces 122(2014):846-850]。以上两种方法均需要过量的酶催化，且Sortase A介导的固定化率较低，一般在30％左右[Hata,Yuto,et al."C‐Terminal‐oriented Immobilization of Enzymes Using Sortase A‐mediated Technique."Macromolecular bioscience 15.10(2015):1375-1380；Ito,Takaomi,et al."Highly oriented recombinant glycosyltransferases:site-specific immobilization of unstable membrane proteins by using Staphylococcus aureus sortase A."Biochemistry 49.11(2010):2604-2614]。
发明简述
在第一方面，提供了一种SpyCatcher肽修饰的载体，其中所述载体，当未被SpyCatcher肽修饰时，包含能够与氨基(NH 2)反应的基团，其中所述SpyCatcher肽通过氨基与所述基团的反应而共价附着于所述载体，并且与所述载体连接的SpyCatcher肽能够与Spytag肽形成异肽键。在一个实施方案中，所述基团选自环氧基、醛基、酰亚胺、氰酸酯、亚胺碳酸酯和酰肼基。
在一个实施方案中，所述SpyCatcher肽优选包含SEQ ID NO：21所示的氨基酸序列。在一个实施方案中，所述SpyCatcher肽包含SEQ ID NO：1、20、22或23所示的氨基酸序列。
在一个实施方案中，所述载体进一步与一种包含SpyTag肽和目的蛋白的融合蛋白连接，其中所述SpyCatcher肽与所述SpyTag肽形成异肽键，所述异肽键由SpyCatcher肽上相应于SEQ ID NO：1所示氨基酸序列的第56位Lys的Lys残基与SpyTag肽上相应于SEQ ID NO：2的第7位Asp的Asp残基形成。在一个实施方案中，所述目的蛋白是酶。在一个实施方案中，所述SpyTag肽包含SEQ ID NO：2、11-15和28任一所示的氨基酸序列。在一个实施方案中，所述目的蛋白是酶，优选选自戊二酰-7-氨基头孢烷酸酰基转移酶、葡萄糖异构酶、腈水合酶、青霉素酰胺酶、天冬氨酸酶、延胡索酸酶、氨基酰化酶、乳糖酶、天冬氨酸-β-脱羧酶和头孢菌素酰胺酶。
在一个实施方案中，所述载体由无机材料、有机材料或无机材料和有机材料的复合材料制成，其中所述无机材料包括但不限于二氧化硅、金属氧化物、粘土材料，所述有机材料包括但不限于琼脂糖、壳聚糖、藻酸盐、明胶、聚丙烯酸、聚丙烯酸酯、聚苯乙烯、聚酰胺、聚丙烯腈，优选所述载体是环氧树脂或氨基树脂。
在一个实施方案中，提供了一种SpyCatcher肽修饰的环氧树脂载体，其中所述SpyCatcher肽通过氨基与环氧基的反应而共价附着于所述载体，优选地，所述环氧树脂载体由聚丙烯酸酯制成。
在一个实施方案中，所述环氧树脂的粒度为100-1000μm，和/或所述环氧树脂的环氧基含量50-800μmol/g wet。
在第二方面，提供了一种制备SpyCatcher肽修饰的载体的方法，包括：
(1)提供包含能够与氨基(NH 2)反应的基团的载体，所述基团优选选自环氧基、醛基、酰亚胺、氰酸酯、亚胺碳酸酯和酰肼基；
(2)在液体环境中将SpyCatcher肽与所述载体接触；任选地，在接触之前将所述基团活化，和/或在接触后，封闭所述载体；以及
(3)任选地，分离SpyCatcher肽修饰的载体。
在第三方面，提供了一种固定化蛋白质的方法，包括：
(1)提供第一方面所述的SpyCatcher肽修饰的载体或根据第二方面所述的方法制备的SpyCatcher肽修饰的载体；
(2)提供包含SpyTag肽和目的蛋白的融合蛋白；
(3)将步骤(1)所述的载体与步骤(2)所述的融合蛋白在允许所述SpyCatcher肽与所述SpyTag肽形成异肽键的条件下相接触，任选地，在接触后，用洗涤缓冲液洗涤去除未固定化的融合蛋白；以及
(4)任选地，分离与所述融合蛋白连接的载体。
在一个实施方案中，所述方法可以用于获得均一化的酶固定化载体。
在一个实施方案中，所述SpyTag肽包含SEQ ID NO：21、22或23所示的氨基酸序列。在一个实施方案中，所述SpyTag肽包含SEQ ID NO：2、11-15和28任一所示的 氨基酸序列。
在一个实施方案中，步骤(1)包括将SpyCatcher肽与未被SpyCatcher肽修饰的载体孵育，获得SpyCatcher肽修饰的载体；以及任选地，用封闭缓冲液处理SpyCatcher肽修饰的载体。
在一个实施方案中，所述目的蛋白是酶，优选选自戊二酰-7-氨基头孢烷酸酰基转移酶、葡萄糖异构酶、腈水合酶、青霉素酰胺酶、天冬氨酸酶、延胡索酸酶、氨基酰化酶、乳糖酶、天冬氨酸-β-脱羧酶和头孢菌素酰胺酶。
在一个实施方案中，所述载体选自环氧树脂和氨基树脂，优选是由无机材料、有机材料或无机材料和有机材料的复合材料制成的载体，其中所述无机材料包括但不限于二氧化硅、金属氧化物、粘土材料，或者是由有机材料制成的载体，所述有机材料包括但不限于琼脂糖、壳聚糖、藻酸盐、明胶、聚丙烯酸、聚丙烯酸酯、聚苯乙烯、聚酰胺、聚丙烯腈。
在第四方面，提供了试剂盒，其包含载体、SpyCatcher肽以及任选存在的包含SpyTag肽和目的蛋白的融合蛋白，其中所述载体和SpyCatcher肽单独存在或以第一方面所述的由SpyCatcher肽修饰的载体的形式存在。在一个实施方案中，所述目的蛋白是酶。
附图说明
图1：基于SpyTag-SpyCatcher反应的蛋白特异性固定化方法示意图。
图2：蛋白表达质粒图谱：a)SpyCatcher表达质粒图谱；b)SpyTag-RFP表达质粒图谱；c)SpyTag-GA表达质粒图谱。
图3：SpyCatcher、SpyTag-RFP和SpyTag-GA表达及纯化SDS-PAGE图。泳道1-5：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。泳道6、7、8分别为融合蛋白SpyTag-GA、SpyCatcher和SpyTag-RFP。
图4：环氧载体固定化SpyCatcher前后的蛋白SDS-PAGE图。泳道1-4：分别为固定前投入5、10、20、40mg/g载体的SpyCatcher上清液；泳道5-8：分别为固定后投入5、10、20、40mg/g载体的SpyCatcher上清液；泳道9-13：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。
图5：固定化Spy-RFP。a)纯化SpyTag-RFP固定前后SDS-PAGE图，泳道1：固定前SpyTag-RFP上清液；泳道2：本发明方法固定SpyTag-RFP后的上清液；泳道3：封闭对照固定SpyTag-RFP后的上清液；泳道4：环氧固定化方法固定SpyTag-RFP后的上清液；泳道5-9：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。b)纯化SpyTag-RFP固定化前后载体颜色对比，其中1号为用本发明方法固定化的SpyTag-RFP；2号为封闭对照组的SpyTag-RFP；3号为环氧方法固定化的SpyTag-RFP。
图6：直接从大肠杆菌(E.coli)细胞裂解液上清中固定化SpyTag-RFP。a)过表达 SpyTag-RFP的大肠杆菌细胞裂解液上清固定前后SDS-PAGE图，泳道1：固定前SpyTag-RFP细胞裂解上清液；泳道2：本发明方法固定SpyTag-RFP后的细胞裂解上清液；泳道3：封闭对照固定SpyTag-RFP后的细胞裂解上清液；泳道4：环氧方法固定SpyTag-RFP后的细胞裂解上清液；泳道5-9：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。b)过表达SpyTag-RFP的大肠杆菌细胞裂解液上清固定前后荧光PAGE图，泳道1：固定前SpyTag-RFP细胞裂解上清液；泳道2：本发明方法固定SpyTag-RFP后的细胞裂解上清液；泳道3：封闭对照固定SpyTag-RFP后的细胞裂解上清液；泳道4：环氧固定化方法固定SpyTag-RFP后的细胞裂解上清液；泳道5-9：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。c)过表达SpyTag-RFP的大肠杆菌细胞裂解液上清固定前后载体颜色对比图。其中1号为用本发明方法固定化的SpyTag-RFP；2号为封闭对照组固定化的SpyTag-RFP；3号为环氧方法固定化的SpyTag-RFP。
图7：纯化SpyTag-GA固定化前后的上清的SDS-PAGE图。泳道1：固定前SpyTag-GA上清液；泳道2：本发明方法固定SpyTag-GA后的上清液；泳道3：封闭对照固定SpyTag-GA后的上清液；泳道4：环氧固定化方法固定SpyTag-GA后的上清液；泳道5-9：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。
图8：直接从大肠杆菌细胞裂解液上清中固定化SpyTag-GA的SDS-PAGE图。泳道1：固定前SpyTag-GA细胞裂解上清液；泳道2：本发明方法固定SpyTag-GA后的细胞裂解上清液；泳道3：封闭对照固定SpyTag-GA后的细胞裂解上清液；泳道4：环氧固定化方法固定SpyTag-GA后的细胞裂解上清液；泳道5-9：含有牛血清蛋白BSA的蛋白定量标准品，上样浓度依次为31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL。
图9：本发明方法SpyCatcher修饰载体与环氧载体固定化蛋白的固定化效率对比。
图10：本发明方法和环氧方法采用相同载体固定化蛋白的固定化效率对比。a)LX1000-EP；b)LX-107S；c)LX-103B；d)HFA001；e)ECR8204；f)ECR8285。
图11：本发明方法SpyCatcher修饰载体与环氧载体固定化蛋白的活力回收率对比。
图12：本发明方法和环氧方法采用相同载体固定化蛋白的活力回收率对比。a)LX1000-EP；b)LX-107S；c)LX-103B；d)HFA001；e)ECR8204；f)ECR8285。
图13：本发明方法SpyCatcher修饰载体与环氧载体固定化蛋白的相对活力对比。
图14：本发明方法和环氧方法采用相同载体固定化蛋白的相对活力对比。a)LX1000-EP；b)LX-107S；c)LX-103B；d)HFA001；e)ECR8204；f)ECR8285。
发明内容
本发明提供一种蛋白固定化方法，以解决蛋白固定化中，对蛋白和载体种类敏感，需要进行固定化试错实验和载体筛选工作的技术问题。
在第一个方面，本公开提供了一种SpyCatcher肽修饰的载体，其中所述载体，当未被SpyCatcher肽修饰时，包含能够与氨基(NH 2)反应的基团，其中所述SpyCatcher肽通过氨基与所述基团的反应而共价附着于所述载体，并且与所述载体连接的SpyCatcher肽能够与Spytag肽形成异肽键。
如本文所用，所述载体是可以用于固定蛋白质的任何载体，只要其含有能够与氨基(NH 2)反应的基团。所述载体可以由各种材料制成，包括但不限于无机材料、有机材料或无机材料和有机材料的复合材料。在一个实施方案中，所述无机材料包括但不限于硅藻土、高岭石、硅胶、多孔质玻璃、活性碳、碳酸钙、陶瓷、二氧化硅、金属氧化物、粘土材料。在一个实施方案中，所述有机材料包括但不限于琼脂糖、壳聚糖、藻酸盐、明胶、聚丙烯酸、聚丙烯酸酯、聚甲基丙烯酸、聚甲基丙烯酸酯、聚苯乙烯、聚酰胺、聚丙烯腈。在一个优选的实施方案中，所述载体是环氧树脂载体或氨基树脂载体。在一个特别优选的实施方案中，所述载体是由聚丙烯酸(酯)制成的载体，特别是环氧树脂载体。
如本文所用，能够与氨基(NH 2)反应的基团是指在不使用活化剂或者使用活化剂的条件下，能够与氨基反应的基团。本领域已知如何活化基团以使其与氨基反应，例如可参见Hermanson,Greg T.Bioconjugate techniques.Academic press,2013。所述氨基可以是肽N-末端的氨基，或者氨基酸残基的侧链上的氨基，例如Lys的ε-氨基。
所述SpyCatcher肽通过一或多个氨基与所述基团的反应而附着于所述载体。
在一个实施方案中，所述SpyCatcher肽通过一个氨基与所述基团的反应而附着于所述载体。在一个实施方案中，所述SpyCatcher肽通过N端氨基与所述基团的反应而附着于所述载体。在另一个实施方案中，所述SpyCatcher肽通过一个Lys的ε-氨基与所述基团的反应而附着于所述载体。
在一个实施方案中，所述SpyCatcher肽通过多个(即两个或更多个)氨基与所述基团的反应而多点附着于所述载体。所述两个或更多个(例如2、3、4、5、6、7、8、9个)氨基可以是N端氨基和一或多个Lys的ε-氨基，或者两个或更多个Lys的ε-氨基。
在一个实施方案中，所述能够与氨基(NH 2)反应的基团选自环氧基、醛基、酰亚胺、氰酸酯、亚胺碳酸酯和酰肼基。
近年发现，化脓性链球菌(Streptococcus pyogenes)中的纤连蛋白结合蛋白(FbaB)的蛋白连接酶能够在其第117位天冬氨酸(Asp117，相应于本文公开的序列SEQ ID NO：2第7位的Asp)和第31位赖氨酸(Lys31，相应于本文公开的序列SEQ ID NO：1第56位的Lys)间迅速地形成异肽键，这一反应称为Spy化学。将FbaB拆开成SpyCatcher和SpyTag两部分后，SpyCatcher和SpyTag之间可以发生Spy反应，通过异肽键连接成一个蛋白。上述Asp117和Lys31残基对于Spy反应非常重要，例如如果Lys31的ε-NH 2与其它基团反应，则不会发生Spy反应。参见例如Science,2007,318,1625-1628][PNAS,2012；109:690；Angew.Chem.Int.Ed.2010,49,8421-8425,JACS.2011,133,478-485。
Spy化学在蛋白标记、蛋白结合、蛋白拓扑学和蛋白材料等方面得到了广泛的研究(PNAS,2012；109:690；Bioconjugate Chem.2017,28:1544-1551；J.Am.Chem.Soc.2013, 135:13988-13997,Angew.Chem.Int.Ed.2019,58,11097-11104；Angew.Chem.Int.Ed.2014,53:6101-6104；PNAS,2014,111,31,11269-11274,Matter 1,1–17)。SpyTag和SpyCatcher的Spy反应的特异性高、SpyTag(13个氨基酸)和SpyCatcher(115个氨基酸)都比较小，而且SpyCatcher可以通过常规的重组表达技术获得(例如以大肠杆菌为表达宿主，用每L的发酵液可以得到110-150mg的Histag纯化SpyCatcher蛋白)。
本公开基于Spy化学，提供了一种蛋白的固定化方法：包括通过SpyCatcher与载体的基团通过共价键连接，得到SpyCatcher修饰的载体，再通过Spy化学将SpyTag与目的蛋白的融合蛋白固定到SpyCatcher修饰的载体上。本发明人出人意料地发现，在将SpyCatcher肽与载体反应后，Lys31仍能与Asp117形成异肽键，发生Spy反应。
如本文所用，术语“蛋白质”、“肽”、“多肽”和“氨基酸序列”可互换使用，指的是任意长度例如两个或更多个氨基酸残基的聚合物。该术语还包括天然地或人为干预地修饰的氨基酸聚合物；例如形成二硫键、糖基化、酯化、乙酰化、磷酸化或任何其他操作与修饰，如与标签或具有生物活性的组分缀合。本文采用的是常规的单字母或三字母氨基酸残基编码。
如本文所用，术语“氨基酸”或“aa”指天然及合成氨基酸以及以类似于天然氨基酸的方式起作用的氨基酸类似物及氨基酸模拟物。天然氨基酸是由遗传密码编码的氨基酸以及那些稍后经修饰的氨基酸，例如羟基脯氨酸、γ-羧基谷氨酸及O-磷酸丝氨酸。氨基酸类似物指与天然氨基酸具有相同基本化学结构(即与氢、羧基、氨基及R基团结合的α-碳)的化合物。氨基酸模拟物指结构不同于氨基酸的一般化学结构但以类似于天然氨基酸的方式起作用的化学化合物。
如本文所用，SpyCatcher肽是能够与SpyTag肽或其变体发生反应形成异肽键连接(Spy反应)的任何肽或其变体。本领域已知各种SpyCatcher肽及其变体，例如可参见Zakeri,B.,Fierer,J.O.,Celik,E.,Chittock,E.C.,Schwarz-Linek,U.,Moy,V.T.,& Howarth,M.(2012).Peptide tag forming a rapid covalent bond to a protein,through engineering a bacterial adhesin.Proceedings of the National Academy of Sciences,109(12),E690-E697；Keeble,A.H.,Banerjee,A.,Ferla,M.P.,Reddington,S.C.,Anuar,I.N.K.,& Howarth,M.(2017).Evolving accelerated amidation by SpyTag/SpyCatcher to analyze membrane dynamics.Angewandte Chemie International Edition,56(52),16521-16525；Keeble,A.H.,Turkki,P.,Stokes,S.,Anuar,I.N.K.,Rahikainen,R.,
Figure PCTCN2021099144-appb-000001
V.P.,& Howarth,M.(2019).Approaching infinite affinity through engineering of peptide–protein interaction.Proceedings of the National Academy of Sciences,116(52),26523-26533。
在一个实施方案中，所述SpyCatcher肽包含SEQ ID NO：21所示的氨基酸序列或其具有一或多个氨基酸取代的氨基酸序列。在一个实施方案中，所述取代是在SEQ ID NO：21的如下一或多个位置的氨基酸取代：15、40、67、69、75、81、83、86、91。在一个实施方案中，所述取代是选自如下的一或多个取代：K15R，Q40H，A67P，T69E，Q75D，N81D，K83E，K86E，I91T。
在一个实施方案中，所述SpyCatcher肽包含SEQ ID NO：21所示的氨基酸序列， 例如包含SEQ ID NO：1或20所示的氨基酸序列。
在一个实施方案中，所述SpyCatcher肽包含SEQ ID NO：20所示的氨基酸序列并且在SEQ ID NO：20所示氨基酸序列的如下一或多个位置的氨基酸取代：2、9、13、19、37、62、89、91、97、103、105、108和113。在一个实施方案中，所述一或多个氨基酸取代是选自如下的在SEQ ID NO：20所示氨基酸序列的一或多个位置的氨基酸取代：D2T，S9G，Q13P，I19T，K37R，Q62H，A89P，T91E，Q97D，N103D，K105E，K108E，I113T。在一个实施方案中，所述一或多个氨基酸取代是选自如下的在SEQ ID NO：20所示氨基酸序列的一或多个氨基酸取代：D2T，S9G，Q13P，I19T，K37R，Q62H，K105E，I113T。在一个实施方案中，所述SpyCatcher肽包含SEQ ID NO：22或23所示的氨基酸序列。
在一个实施方案中，本发明所述具有一或多个氨基酸取代的SpyCatcher肽可包含与SEQ ID NO：1、20、21、22或23所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高序列相同性的氨基酸序列。
序列相同性可以通过商业可获得的计算机程序来确定，其采用任何适宜的算法计算出两个或多个序列间的相同性百分比，例如采用缺省参数。此类计算机程序的典型的例子是CLUSTAL。更有利地，应用BLAST算法，参数设置为缺省值。在National Center for Biotechnology Information(NCBI)网站上有BLAST算法的详细介绍。
在一个实施方案中，所述载体是环氧载体或氨基载体，即在载体上存在环氧基团或氨基，例如是环氧树脂和氨基树脂。由于环氧基的化学活性，可用多种含有活泼氢的化合物使其开环。在共价载体中，环氧载体几乎是理想的基质，可以在实验室和工业规模上非常容易地固定蛋白质。在非常温和的实验条件(例如pH 7.0)下，环氧载体与蛋白质发生反应。在一个实施方案中，所述载体是由无机材料、有机材料或无机材料和有机材料的复合材料制成的载体，其中所述无机材料例如包括但不限于二氧化硅、金属氧化物、粘土材料，所述有机材料例如包括但不限于琼脂糖、壳聚糖、藻酸盐、明胶、聚丙烯酸、聚丙烯酸酯、聚甲基丙烯酸、聚甲基丙烯酸酯、聚苯乙烯、聚酰胺、聚丙烯腈。
如本文所用，环氧树脂是指含有环氧基的树脂，氨基树脂是指含有氨基的树脂，其中所述环氧基和氨基可以是在树脂上通过修饰得到的环氧基和氨基。
如本文所用，环氧树脂是指含有环氧基的聚合物，可包括例如环氧树脂、氨基环氧树脂、羧基环氧树脂、巯基-二硫键环氧树脂。环氧树脂例如包括但不限于Lifetech TM ECR8285、ECR8204、ECR8209、LX1000EA、LX1000EP、LX103B、EP200、LX1000HFA、HFA001、LX107S、LX1000SW、LX1000SD、
Figure PCTCN2021099144-appb-000002
C、
Figure PCTCN2021099144-appb-000003
C250L、
Figure PCTCN2021099144-appb-000004
FP-EC3、
Figure PCTCN2021099144-appb-000005
EC-EP/M、
Figure PCTCN2021099144-appb-000006
EC-Ep、ES1、ES103、ES105、ES108和ES109。
在一个实施方案中，所述环氧树脂的粒度为约5-2000μm，例如约10-2000、20-2000、30-2000、40-2000、50-2000、50-1900、50-1800、50-1700、50-1600、50-1500、50-1400、50-1300、50-1200、50-1100、50-1000、50-900、50-800、50-700、50-600、50-500、50-450、50-400、50-350、100-450、100-400、100-350、100-300、150-450、150-400、150-350、 150-300μm，如约5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、350、400、450、500、600、700、800、900、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000μm。如本文所用，所述粒度是指颗粒的平均直径(中位值(D50))，用μm表示。
在一个实施方案中，所述环氧树脂的环氧基含量少于1000μmol/g wet，如约10-1000μmol/g wet，例如约10-900、10-800、10-700、10-600、10-500、10-400、10-300、10-200、10-150、10-100、20-900、20-800、20-700、20-600、20-500、20-400、20-300、20-200、20-150、20-100、30-900、30-800、30-700、30-600、30-500、30-400、30-300、30-200、30-150、30-100、40-900、40-800、40-700、40-600、40-500、40-400、40-300、40-200、40-150、40-100、50-900、50-800、50-700、50-600、50-500、50-400、50-300、50-200、50-150、50-100、50-90、50-80、70-90、80-100、75-95、390-520、585-780μmol/g wet，如约10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、400、450、500、550、600、650、700、750、800、850、900、950、1000μmol/g wet。如本文所用，所述环氧基含量是指每g湿树脂上环氧基团的摩尔数，用μmol/g wet表示。
在一个实施方案中，所述环氧树脂是亲水性的。
在一个实施方案中，所述环氧树脂由聚丙烯酸酯制成，例如聚丙烯酸甲酯、乙酯、丙酯、丁酯、异丁酯、叔丁酯等。在一个实施方案中，所述环氧树脂由聚丙烯酸甲酯制成。在一个实施方案中，所述环氧树脂由聚丙烯酸乙酯制成。在一个实施方案中，所述环氧树脂由聚丙烯酸丙酯制成。在一个实施方案中，所述环氧树脂由聚丙烯酸丁酯制成。在一个实施方案中，所述环氧树脂由聚丙烯酸异丁酯制成。在一个实施方案中，所述环氧树脂由聚丙烯酸叔丁酯制成。
在一个实施方案中，所述环氧树脂上与Spycatcher肽连接的基团是环氧基。
在一个实施方案中，提供了一种如本文所述的SpyCatcher肽修饰的环氧树脂载体，其中所述SpyCatcher肽通过氨基与树脂的环氧基反应而共价附着于所述载体。优选地，所述环氧树脂载体由聚丙烯酸酯制成。更优选地，所述环氧树脂的粒度为约100-350μm，和/或所述环氧树脂的环氧基含量约50-100μmol/g wet。
如本文所用，氨基(NH 2)树脂是指含有氨基的聚合物，可包括例如含有氨基的聚丙烯酸酯、含有氨基的聚甲基丙烯酸酯、含有氨基的聚苯乙烯等。氨基树脂例如包括但不限于LX-1000EA，LX-1000HA，ECR8305，ECR8309，ECR8315，ECR8404，ECR8409，ECR8415。
在第二个方面，提供了制备SpyCatcher肽修饰的载体的方法，包括：
(1)提供包含能够与氨基(NH 2)反应的基团的未修饰的载体，所述基团优选选自环氧基、醛基、酰亚胺、氰酸酯、亚胺碳酸酯和酰肼基；
(2)在液体环境中将SpyCatcher肽与所述载体接触；任选地，在接触之前将所述 基团活化，和/或在接触后，封闭所述载体；以及
(3)任选地，分离SpyCatcher肽修饰的载体。
所述载体、SpyCatcher肽等如前所述。
本文所述的未修饰的载体可以通过本领域已知的任何途径获得。在与SpyCatcher肽接触前，所述载体可以使用活化剂活化，或者可以直接与SpyCatcher肽接触(不使用活化剂)。反应条件是本领域已知的或者可以根据本领域已知技术容易地确定。
如本文所用，液体环境是指适合于所述SpyCatcher肽通过肽上的氨基(例如N端和侧链氨基，或者侧链氨基)与所述能够与氨基反应的基团反应而共价附着于所述载体的液体环境，例如PBS缓冲液(如0.1M，pH 7.0)。例如可参见Zakeri,Bijan,et al."Peptide tag forming a rapid covalent bond to a protein,through engineering a bacterial adhesin."Proceedings of the National Academy of Sciences 109.12(2012):E690-E697。
如本文所用，活化是指对基团进行处理以使其能够与SpyCatcher肽上的氨基反应，例如使用戊二醛作为活化剂活化载体上的氨基。例如可参见Walt,David R.,and Venetka I.Agayn."The chemistry of enzyme and protein immobilization with glutaraldehyde."(1994):425-430；Immobilization of enzymes and cells.Humana Press,2006。
在一个实施方案中，第二个方面所述方法步骤(2)包括将SpyCatcher肽与未修饰的载体孵育，例如至少约1、2、3、4、5、6、7、8、9、10、11、12、24、36、48小时，获得SpyCatcher肽修饰的载体；以及任选地，用封闭缓冲液处理SpyCatcher肽修饰的载体，例如处理至少约1、2、3、4、5、6、7、8、9、10、11、12、24、36、48小时。在一个实施方案中，所述孵育在约20-50rpm例如约25-40、25-35、25-30rpm转速下离心反应。封闭完成后，可以分离修饰的载体，例如通过离心去除封闭缓冲液。在一个实施方案中，用于孵育的SpyCatcher肽与未修饰的载体的比例为10-40mg肽/g载体，例如10、15、20、25、30、35、40mg肽/g载体，特别优选20mg肽/g载体。
第二个方面所述方法步骤(2)可以在任何适合的温度、pH下进行。例如，所述温度可以是约20-37℃，如约20-35℃，25-37℃，25-35℃，20-30℃，25-35℃，特别是约20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37℃。所述pH值可以任何合适的pH，例如约4.0-10.0，如约5.0-9.0、6.0-8.0或6.0-7.0，特别是约4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.0。
如本文所用，术语“封闭”是指将剩余的未与SpyCatcher发生反应的基团例如环氧基团反应完，其目的在于使所述基团在第二步的固定化过程中不再与目的蛋白发生反应。本文所述封闭缓冲液是本领域常用的封闭缓冲液，本领域已知对于本文所述基团的合适的封闭缓冲液(例如可参见Hermanson,Greg T.Bioconjugate techniques.Academic press,2013；Immobilization of enzymes and cells.Humana Press,2006)。例如，本文所述封闭缓冲液是本领域常用的环氧基团封闭缓冲液，如甘氨酸、乙醇胺、牛血清白蛋白等。在一个实施方案中，所述封闭缓冲液包含但不限于：甘氨酸，pH 8.5；乙醇胺，pH 8.5。在一个实施方案中，所述封闭缓冲液是3M甘氨酸，pH8.5。
第二个方面所述方法步骤(3)所述的分离SpyCatcher肽修饰的载体，可以采用本 领域已知的任何合适的技术进行分离，例如离心。
在进一步的实施方案中，本文所述的或者根据本文所述方法获得的SpyCatcher肽修饰的载体与包含SpyTag肽和目的蛋白的融合蛋白连接，其中所述SpyCatcher肽与所述SpyTag肽形成的异肽键(Spy反应)，从而将所述目的蛋白与所述SpyCatcher肽修饰的载体连接。
如前所述，SpyTag肽也是本领域已知的可以发生Spy反应的任何肽。本领域已知各种产生融合蛋白的技术和手段。在融合蛋白中，所述SpyTag肽可以位于融合蛋白的N端、C端或中间的任何位置，只要所获得的融合蛋白具有所需的功能活性即可。本领域技术人员根据本领域技术知识可以容易地检测蛋白质的功能活性，这均在本领域技术人员的技术能力范围内。
在一个实施方案中，本文所述SpyTag肽包含SEQ ID NO：2、11、12、13、14、15或28任一所示的氨基酸序列。
在一个实施方案中，所述肽SpyTag位于融合蛋白的N端或C端。
在一个实施方案中，本发明所述融合蛋白中的SpyTag肽和目的蛋白可以通过接头连接，例如SpyTag-接头-目的蛋白或目的蛋白-接头-SpyTag。
如本文所用，所述接头是连接SpyTag肽和目的蛋白的肽或其他分子。所述连接可以通过本领域已知的连接两个部分的任何方法连接，只要接头部分不明显妨碍所述融合蛋白中蛋白质的所需功能活性和/或不明显妨碍SpyTag肽与SpyCatcher肽的Spy反应即可。本领域技术人员可以根据本领域技术知识容易地确定和选择合适的接头。
如本文所用，本发明所述的SpyTag肽与目的蛋白的融合蛋白，包含SpyTag肽与目的蛋白，还可以包含多肽接头与亲和标签。在一个实施方案中，所述多肽接头位于SpyTag与目的蛋白之间。
如本文所用，“亲和标签”是指在融合蛋白的N端或C端添加的亲和标签，便于后续目的蛋白的纯化。所述SpyTag与目的蛋白的融合蛋白可以是纯化的融合蛋白，也可以是未纯化的融合蛋白，如细胞裂解液。在一个实施方案中，所述SpyTag的氨基酸序列如SEQ ID NO：2所示。所述SpyTag肽可以在目的蛋白的C端或N端。
在一个实施方案中，所述接头长度优选不少于9个氨基酸残基，例如9、11、13、15个氨基酸残基或更长。本领域技术人员已知的任何接头均可用于本发明中。接头部分可以是肽。用于接头的典型氨基酸残基是甘氨酸、丝氨酸、酪氨酸、半胱氨酸、赖氨酸、谷氨酸和天冬氨酸等等。举例的这种已知的接头部分包括但不限于(GGGGS) 3(SEQ ID NO：27)、(G) nS(G) n，其中n＝4、5、6或7。在一个实施方案中，所述接头为(GGGGS) 3。
在本文中，与SpyTag连接的目的蛋白可以是任何希望固定在载体之上的蛋白质，包括例如但不限于酶、辅因子、伴侣蛋白等。在一个实施方案中，所述目的蛋白是酶，例如是本领域已知的可以固定在本文所述载体上的任何酶，并且所述酶可以来自任何合适的来源，其可以是分离自天然来源例如细菌或者人工合成例如通过重组技术表达的。
在一个实施方案中，所述酶选自葡萄糖异构酶(EC 5.3.1.5)、腈水合酶(EC 4.2.1.84)、青霉素酰胺酶(EC 3.5.1.11)、天冬氨酸酶(EC 4.3.1.1)、延胡索酸酶(EC 4.2.1.2)、氨 基酰化酶(EC 3.5.1.14)、乳糖酶(EC 3.2.1.108)、天冬氨酸-β-脱羧酶(EC 4.1.1.12)和头孢菌素酰胺酶(EC 3.5.1.11)。
在一个实施方案中，本公开提供了一种SpyCatcher肽修饰的环氧树脂载体，其中所述SpyCatcher肽通过氨基与载体的环氧基反应而共价附着于所述载体。在一个实施方案中，所述SpyCatcher肽具有SEQ ID NO：1所示的氨基酸序列。在一个实施方案中，所述环氧树脂由聚丙烯酸酯制成。优选地，所述环氧树脂的粒度为约100-350μm，和/或所述环氧树脂的环氧基含量约50-100μmol/g wet。
在一个实施方案中，本公开提供了一种固定了蛋白质的环氧树脂载体，所述环氧树脂与具有SEQ ID NO：1所示氨基酸序列的SpyCatcher肽连接，所述蛋白质是包含SEQ ID NO：2所示氨基酸序列的SpyTag肽和目的蛋白的融合蛋白。优选地，所述环氧树脂由聚丙烯酸酯制成。
在第三个方面，本公开提供了一种固定化蛋白质的方法，包括：
(1)提供上述第一个方面所述的SpyCatcher肽修饰的载体或者根据第二个方面的方法获得的SpyCatcher肽修饰的载体；
(2)提供包含SpyTag肽和目的蛋白的融合蛋白；以及
(3)将步骤(1)所述的载体与步骤(2)所述的融合蛋白在允许所述SpyCatcher肽与所述SpyTag肽形成异肽键的条件下相接触，任选地，在接触后，用洗涤缓冲液洗涤去除未固定化的融合蛋白；以及
(4)任选地，分离与所述融合蛋白连接的载体。
所述载体、SpyCatcher肽、SpyTag肽、蛋白质等如前所述。
在一个实施方案中，第三个方面所述方法步骤(1)包括将SpyCatcher肽与未修饰的载体孵育，例如至少约1、2、3、4、5、6、7、8、9、10、11、12、24、36、48小时，获得SpyCatcher肽修饰的载体；以及任选地，用封闭缓冲液处理SpyCatcher肽修饰的载体，例如处理至少约1、2、3、4、5、6、7、8、9、10、11、12、24、36、48小时。在一个实施方案中，所述孵育在约20-50rpm例如约25-40、25-35、25-30rpm转速下离心反应。封闭完成后，可以分离修饰的载体，例如通过离心去除封闭缓冲液。在一个实施方案中，用于孵育的SpyCatcher肽与未修饰的载体的比例为10-40mg肽/g载体，例如10、15、20、25、30、35、40mg肽/g载体，特别优选20mg肽/g载体。
第三个方面所述方法步骤(1)可以在任何适合的温度、pH下进行。例如，所述温度可以是约20-37℃，如约20-35℃，25-37℃，25-35℃，20-30℃，25-35℃，特别是约20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37℃。所述pH值可以任何合适的pH，例如约4.0-10.0，如约5.0-9.0、6.0-8.0或6.0-7.0，特别是约4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.0。
第三个方面所述方法步骤(2)中所述与SpyTag肽连接的目的蛋白可以是分离纯化的蛋白质，例如经本领域已知技术分离纯化的融合蛋白；也可以是含有所述肽和目的蛋白的融合蛋白的未经进一步纯化的细胞裂解液，例如表达融合蛋白的细胞裂解液。
所述融合蛋白可以通过在原核生物、酵母或高等真核生物细胞中重组表达获得，示 例性原核生物包括埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、沙门氏菌属(Salmonella)以及假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)的细菌。在优选的实施方案中，重组细胞是埃希氏菌属细胞，优选大肠杆菌(Escherichia coli)。在本发明的一个具体实施方案中，所使用的重组细胞为大肠杆菌BL21(DE3)菌株细胞(Novagen)。本发明所述的目的蛋白可以是任何蛋白，如本发明实施例中所列举的红色荧光蛋白(RFP)、戊二酰-7-氨基头孢烷酸酰基转移酶(GA)。
所述细胞裂解液可以通过本领域已知方法获得，例如破碎细胞的方法包括但不限于：超声、匀浆、高压(例如在弗氏压碎器中)、低渗(osmolysis)、去垢剂、裂解酶、有机溶剂或其组合。在一个实施方案中，所述破碎在第一pH条件(即弱碱性pH，例如pH7.2-8.5，优选pH 7.4)下进行，由此使得宿主细胞的细胞膜破碎，蛋白上清从破碎后菌体中释放出来，且仍然保持可溶状态。所述纯化的融合蛋白，可以通过亲和标签纯化获得。在一些实施方案中，所述亲和标签为常见的6×His标签，位于目的蛋白的C端。释放出来的蛋白上清通过蛋白亲和层析方式纯化。重组细胞的细胞膜破碎后，可以通过离心收集上清，除去不可溶的沉淀部分，然后通过融合蛋白的His标签进行亲和层析纯化。
在第三个方面所述方法步骤(3)中，所述修饰的载体和所述融合蛋白在允许SpyCatcher肽与SpyTag肽形成异肽键的条件下接触。本领域已知允许SpyCatcher肽与SpyTag肽形成异肽键(发生Spy反应)的条件(如前所述)。
如本文所用，接触是将所述修饰的载体和所述蛋白质物理性相关，例如在溶液中混合两种物质，或者通过向含有一种物质(例如融合蛋白)的溶液中加入另一种物质(例如修饰的载体)而进行。
在一个实施方案中，第三个方面所述方法步骤(3)包括将SpyCatcher修饰的载体与所述融合蛋白接触例如至少约1、2、3、4、5、6、7、8、9、10、11、12、24、36、48小时。在一个实施方案中，所述接触在约20-50rpm例如约25-40、25-35、25-30rpm转速下离心反应。
第三个方面所述方法步骤(3)可以在任何适合的温度、pH下进行。例如，所述温度可以是约4-37℃，如约5-35℃、10-35℃、15-35℃、20-35℃，25-37℃，25-35℃，20-30℃，25-35℃，特别是约4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37℃。所述pH值可以任何合适的pH，例如约4.0-10.0，如约5.0-9.0、6.0-8.0或6.0-7.0，特别是约4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.0。在一个实施方案中，pH6.0-7.0是特别优选的。
在一个实施方案中，第三个方面所述方法步骤(3)包括将SpyTag肽与目的蛋白的融合蛋白与步骤(1)获得的与SpyCatcher肽连接的载体孵育；以及任选用洗涤缓冲液洗涤去除未固定化的融合蛋白。
如本文所用，所述洗涤缓冲液是本领域常用的蛋白洗涤缓冲液，如磷酸盐缓冲液(PBS)、Tris-HCl缓冲液等。
第三个方面所述方法步骤(4)所述的分离与所述融合蛋白连接的载体，可以采用 本领域已知的任何合适的技术进行分离，例如离心。
在一个实施方案中，提供了一种基于Spy反应的蛋白固定化方法，所述方法包括：
-将SpyCatcher肽与环氧载体孵育，获得SpyCatcher肽修饰的载体；
-用封闭缓冲液处理SpyCatcher肽修饰的载体；
-将SpyTag肽和目的蛋白的融合蛋白与SpyCatcher肽修饰的载体孵育；
-用洗涤缓冲液洗涤去除未固定化的融合蛋白；以及
-任选地，分离与所述融合蛋白连接的载体。
在第四个方面，提供了一种试剂盒，其包含载体、SpyCatcher肽以及任选存在的SpyTag肽和目的蛋白的融合蛋白。
所述载体、SpyCatcher肽、SpyTag肽、蛋白质等如前所述。
在一个实施方案中，所述载体和SpyCatcher肽单独存在。在使用前，可以将载体和肽相接触(例如在溶液中混合)，制备第一个方面所述的SpyCatcher肽修饰的载体。
在另一个实施方案中，所述载体和SpyCatcher肽以第一个方面所述的SpyCatcher肽修饰的载体的形式存在。
除非上下文另有指示，词语“或”旨在包括“和”。
如本文所用，“任选”或“任选地”是指随后描述的事件或情况发生或不发生，该描述包括其中所述事件或情况发生及不发生的情况。例如，任选包括的步骤是指该步骤存在或不存在。
如本文所用，术语“约”是指包括具体数值的数值范围，本领域技术人员可以合理认为其类似于具体数值。在某些实施方案中，术语“约”是指在使用本领域通常接受的测量的标准误差内。例如，在某些实施方案中，约是指到具体数值的+/-10％或5％。
如本文所用，当说明书中针对一个特征列出具体数值或比例时，也涵盖了任意其两个数值或比例组成的范围。例如列出数值1、2、3、4时，也涵盖了1-2、1-3、1-4、2-3、2-4和3-4。
本公开提供的方法与现有的商业固定化载体兼容，不需要新的载体表面化学，不需要额外的酶催化剂如sortase，并且可以直接使用含有所需蛋白质的细胞裂解液进行固定化。本公开方法的固定化过程(在环氧载体情况下)只需要1步，连接反应特异，效率高，条件温和(室温、中性pH)，不需要添加额外的化学试剂。本公开方法改良的固定化载体通用性好，其固定化效率与活力回收率高，均一性好，可减少对固定化载体的筛选的工作。
具体实施方式
以下通过实例对本发明的具体实施方式作进一步的说明，但本发明的实施方式不限于此。以下实施例中所用方法如无特别说明均为常规方法，具体步骤可参见，例如，《Molecular Cloning:A Laboratory Manual》(Sambrook,J.,Russell,David W.,Molecular Cloning:A Laboratory Manual,3 rd edition,2001,NY,Cold Spring Harbor)。所用引物和测序均由生工生物工程(上海)股份有限公司合成。
实施例1：构建蛋白表达质粒
本发明中使用的寡聚核苷酸引物如表1所示。
表1 本发明使用的寡聚核苷酸引物序列
Figure PCTCN2021099144-appb-000007
a引物下划线部分分别为限制性内切酶Nde Ⅰ、Hind III、Sac Ⅰ和Xho I的识别位点。
1.1构建SpyCatcher的表达质粒
本发明实施例中所构建的pET30a(+)-PT linker-SpyCatcher-His表达质粒(SEQ ID NO：17)用于表达Ptlingker-SpyCatcher-His蛋白，质粒图谱如图2a所示。
首先以质粒pET-30a(+)-FS4-SpyCatcher(SEQ ID NO：9)为DNA模板，利用表1中的引物SpyCatcher-F和SpyCatcher-R进行PCR扩增，获得Nde Ⅰ-PT linker-SpyCatcher-Xho I多核苷酸片段。PCR反应使用Q5高保真DNA聚合酶(NEB，M0491)，PCR体系如表2所示。
表2 PCR反应体系和程序
Figure PCTCN2021099144-appb-000008
PCR反应程序：a)98℃ 30sec；b)98℃ 10sec；c)57℃ 15sec；d)72℃ 15sec；e)步骤b-d，30循环；f)72℃ 2min；g)4℃ hold。
PCR反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，结果PCR扩增 出与预期相符的目标DNA条带。使用超薄DNA产物纯化试剂盒(天根生化科技(北京)有限公司，DP203-02)纯化回收目标DNA，-20℃保存备用。
将纯化回收的目标DNA用限制性内切酶Nde Ⅰ(NEB，RO111L)和Xho I(NEB，R0146L)进行双酶切后，与经同样酶双酶切的质粒pET-30a(+)(Novagen)进行连接，将连接产物转化到大肠杆菌BL21(DE3)(Novagen)感受态细胞，将转化细胞涂布于添加有50μg/mL卡那霉素(Kanamycin,Kan)的LB(Luria-Bertani培养基)平板上，选择阳性克隆，提取质粒，对其进行测序，测序结果表明所克隆的pET30a(+)-PT linker-SpyCatcher-His序列正确。
1.2构建SpyTag融合蛋白的表达质粒
本发明实施例中所构建的pET30a(+)-SpyTag-GS linker-RFP-His(SEQ ID NO：18)表达质粒用于表达SpyTag与红色荧光蛋白(RFP)的融合蛋白SpyTag-RFP(SEQ ID NO：24)，其质粒图谱如图2b所示；pET30a(+)-SpyTag-GS linker-GA-His表达质粒用于表达SpyTag与戊二酰-7-氨基头孢烷酸酰基转移酶(glutaryl-7-aminocephalosporanic acid acylase,GA)的融合蛋白SpyTag-GA(SEQ ID NO：25)，其质粒图谱如图2c所示。
1)构建质粒pET30a(+)-SpyTag-Gslinker-RFP-His：首先以质粒pET30a(+)-Spy-RFP(见SEQ ID NO：10)作为DNA模板，利用如表1中的引物RFP-F和RFP-R进行PCR扩增获得Nde Ⅰ-SpyTag-Gslinker-RFP-Hind Ⅲ多核苷酸片段。PCR反应使用Q5高保真DNA聚合酶，PCR体系如表3所示。
表3 PCR反应体系
Figure PCTCN2021099144-appb-000009
PCR反应程序：a)98℃ 30sec；b)98℃ 10sec；c)57℃ 20sec；d)72℃ 30sec；e)步骤b-d，30循环；f)72℃ 2min；g)4℃ hold。
反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，结果PCR扩增出与预期相符的目标DNA条带。使用超薄DNA产物纯化试剂盒纯化回收目标DNA，-20℃保存备用。
将纯化回收的目标DNA用限制性内切酶Nde Ⅰ和Hind Ⅲ(NEB，R3104L)进行双酶切后，与经同样酶双酶切的质粒pET-30a(+)进行连接，将连接产物转化到 Ecoli.BL21(DE3)感受态细胞，将转化细胞涂布于添加有50μg/mL Kan的LB平板上，选择阳性克隆，提取质粒，对其进行测序，测序结果表明所克隆的pET30a(+)-SpyTag-Gslinker-RFP-His序列正确。
2)构建质粒pET30a(+)-SpyTag-Gslinker-GA-His(SEQ ID NO：19)：假单胞菌SY-77(Pseudomonas sp.SY-77)的GA编码序列(SEQ ID NO:16)由生工生物科技有限公司(上海)经密码子优化后合成，GA的蛋白序列如SEQ ID NO:26所示。利用如表1中的引物GA-F和GA-R进行PCR扩增获得Sac Ⅰ-GA-Hind Ⅲ多核苷酸片段。PCR反应使用Q5高保真DNA聚合酶，PCR体系和程序如表4所示。
表4 PCR反应体系
Figure PCTCN2021099144-appb-000010
PCR反应程序：a)98℃ 30sec；b)98℃ 10sec；c)57℃ 20sec；d)72℃ 90sec；e)步骤b-d，30循环；f)72℃ 4min；g)4℃ hold。
反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，结果PCR扩增出与预期相符的目标DNA条带。使用超薄DNA产物纯化试剂盒纯化回收目标DNA，-20℃保存备用。
将纯化回收的目标DNA用限制性内切酶Sac Ⅰ(NEB，R0156L)和Hind Ⅲ进行双酶切后，与经同样酶双酶切的质粒pET30a(+)-SpyTag-Gslinker-GA-His进行连接，将连接产物转化到大肠杆菌BL21(DE3)感受态细胞，将转化细胞涂布于添加有50μg/mL Kan的LB平板上，选择阳性克隆，提取质粒，对其进行测序，测序结果表明所克隆的pET30a(+)-SpyTag-Gslinker-GA-His序列正确。
本领域技术人员通过常规操作可以制备这些质粒。
实施例2：融合蛋白的表达、纯化和活性测定
2.1融合蛋白的表达
将实施例1中构建的菌株(含有质粒pET30a(+)-PT linker-SpyCatcher-His)按1:50的接种量接种到含50μg/mL卡那霉素(Kan)的LB液体培养基中，过夜培养后，转接 到新鲜的LB(50μg/mL Kan)培养基中，并在37℃摇床中培养至对数期(OD 600＝0.4-0.6)，加入异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside,IPTG)至终浓度为0.2mM，在30℃诱导6小时，测量菌浓度OD 600，收获细胞。
将实施例1中构建的菌株(含有质粒pET30a(+)-SpyTag-GS linker-RFP-His)接种到含50μg/mL Kan的LB液体培养基中，按1:50的接种量，将过夜培养的菌液转接到新鲜的LB(50μg/mL Kan)培养基中，并在37℃摇床中培养至对数期(OD 600＝0.4-0.6)，加入IPTG至终浓度为0.2mM，在23℃诱导22小时，测量菌浓度OD 600，收获细胞。
将实施例1中构建好的菌株(含有质粒pET30a(+)-SpyTag-GS linker-GA-His)接种到含50μg/mL Kan的LB液体培养基中，按1:50的接种量，将过夜培养的菌液转接到新鲜的LB(50μg/mL Kan)培养基中，并在37℃摇床中培养至对数期(OD 600＝0.4-0.6)，加入IPTG至终浓度为0.1mM，在18℃下诱导12小时，测量菌浓度OD 600，收获细胞。
2.2 SDS-PAGE检测和定量
收获步骤2.1所得的细胞，用PBS缓冲液(0.1M，pH 7.0)重悬至50OD/mL。在冰上通过超声破碎细胞(破碎条件为：功率200W，超声时间3sec，间隔时间3sec，超声次数99次)。超声完成后，通过离心分离缓冲液的上清和沉淀。为了尽可能去除沉淀中混杂的可溶成分，用等体积的缓冲液将得到的沉淀洗涤两遍。上清液和沉淀重悬液直接用于SDS-PAGE测定。以梯度浓度的BSA(31.25μg/mL,62.5μg/mL,125μg/mL,250μg/mL,500μg/mL)作为定量标准，采用ImageJ软件(National Institutes of Health，美国)进行定量。
2.3融合蛋白的纯化及透析
收获实施案例2.1中的细胞，用PBS缓冲液(0.1M，pH 7.0)重悬至50OD/mL。用高压匀浆机(PhD Technology International LLC,USA)破碎细胞(破碎条件为：压强12000psi，循环破碎3次)。高压匀浆破碎完成后，通过离心分离裂解液的上清和沉淀。收集上清，进行镍柱亲和层析纯化，收集纯化前后及流穿液样品，SDS-PAGE检测纯化效果。
将纯化后的蛋白上清进行透析，透析缓冲液为PBS缓冲液(0.1M，pH 7.0)。
SDS-PAGE的分析结果如图3所示。泳道6、7、8分别为融合蛋白SpyTag-GSlinker-GA(为了简便，以下称为SpyTag-GA)、PT linker-SpyCatcher(为了简便，以下称为SpyCatcher)和SpyTag-Gslinker-RFP(为了简便，以下称为SpyTag-RFP)，纯化后三种融合蛋白的纯度分别为95％、81％、88％，可用于后续的固定化实验。
2.4融合蛋白的酶活和荧光测定
收获步骤2.1所得的细胞，用PBS缓冲液(0.1M，pH 7.0)重悬50OD/mL。用高压匀浆机(PhD Technology International LLC,USA)破碎细胞(破碎条件为：压强12000psi，循环破碎3次)。高压匀浆破碎完成后，通过离心分离裂解液的上清和沉淀。上清液直接用于相应的酶活和荧光测定。GA的酶活和RFP的荧光测定方法如下：
GA酶活力测定原理：GA催化戊二酰-7-氨基头孢烷酸(GL-7-ACA)水解为7-氨基头孢烷酸(7-ACA)。7-ACA的伯氨基与对二甲胺基苯甲醛(pDAB)反应，生成黄色席夫碱， 在415nm处具有最大吸光度。
游离GA酶活力测定：将20μL酶液(在pH 7.0的0.1M PBS缓冲液中)与20μL底物(在pH 7.0的0.1M PBS缓冲液中的1％(w/v)GL-7-ACA)混合，在37℃下孵育10分钟。加入140μL 20％(v/v)乙酸和0.05M NaOH的混合物(2：1，v/v)终止反应，然后加入20μL 0.5％(w/v)pDAB。将该混合物在25℃下再孵育10分钟，然后测量在415nm的吸光度。一个GA活性单位(U)定义为在37℃，pH 7.0下每分钟产生1μmol 7-ACA所需的酶量。
游离GA酶活力计算：
Figure PCTCN2021099144-appb-000011
ΔA/min—表示每分钟吸光度的变化值，即为斜率；
S—7-ACA的摩尔消光系数(1.576cm 2/μmoL)，通过标准曲线求得；
d—光程(反应体系为0.2mL，光程约为0.5cm)；
Vt—表示反应液的总体积(0.2mL)；
Vs—表示样品酶液的体积(0.02mL)；
X—表示样品酶液的稀释倍数。
游离RFP荧光检测：纯化的RFP适当稀释后，取200μL加入到黑色的平底96孔板(Corning，3925)中，使用光栅型多功能酶标仪(infinite M200PRO，TECAN)在37℃下检测RFP荧光强度，其激发波长为588nm，发射波长为635nm，增益值为100％[Anuar I N A K,Banerjee A,Keeble A H,et al.Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox[J].Nature communications,2019,10(1):1734.]。
实施例3：制备SpyCatcher修饰载体
本发明首先获得SpyCatcher修饰载体，然后通过Spy化学一步固定化SpyTag融合蛋白。
3.1 SpyCatcher修饰载体的制备
分别称取50mg环氧载体LX-1000EP(西安蓝晓科技新材料股份有限公司)，用PBS缓冲液(0.1M，pH 7.0)将LX-1000EP洗涤3次，分别加入0.25、0.5、1或2mg纯化的SpyCatcher，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应12h。
同时取等量的SpyCatcher上清作为空白对照并检测其蛋白量。
反应完成后，通过离心去除上清，用PBS缓冲液(0.1M，pH 7.0)洗涤5次，获得SpyCatcher修饰载体(为了简便，以下称为SpyCatcher-LX-1000EP)。计算SpyCatcher的固定量及固定化效率。相关计算公式如下：
SpyCatcher固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000012
SDS-PAGE的分析结果如图4所示。依照蛋白定量标准品，应用ImageJ凝胶定量分析软件对目的条带进行光密度分析，计算得出融合蛋白SpyCatcher的固定化效率(表5)。由表可知，随着SpyCatcher的投入量增加，SpyCatcher的固定化效率随之降低，在投入20mg/g载体时，其固定化效率还能保持在87.5％，与投入10mg/g载体相比未减少很多，反之，当投入量增加到40mg/g载体时，其固定化效率减少到63.7％，由此，本发明其它实验将SpyCatcher的投入量确定为20mg/g载体。
表5 SpyCatcher的固定化结果
Figure PCTCN2021099144-appb-000013
将得到的固定化SpyCatcher-LX-1000EP加入12倍体积的封闭缓冲液(3M甘氨酸，pH 8.5)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应24h。封闭完成后，离心去除封闭液，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，存于4℃。
实施例4：融合蛋白的固定化
4.1不同pH下固定化SpyTag-RFP
采用SpyCatcher-LX-1000EP在不同pH下固定化纯化的SpyTag-RFP。
本发明方法：称取50mg SpyCatcher-LX-1000EP，用0.1M的PBS缓冲液(pH分别为5.0、6.0、7.0、8.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)(pH分别为5.0、6.0、7.0、8.0)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。
封闭对照：称取50mg经封闭反应之后的环氧载体，用0.1M的PBS缓冲液(pH分别为5.0、6.0、7.0、8.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)(pH分别为5.0、6.0、7.0、8.0)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为封闭对照组。
环氧方法：称取50mg LX-1000EP，用0.1M的PBS缓冲液(pH分别为5.0、6.0、7.0、8.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)(pH分别为5.0、6.0、7.0、8.0)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为对照组。
同时取等量的SpyTag-RFP上清作为空白对照并检测其蛋白量。
反应完成后，通过离心分离出固定化RFP。用PBS缓冲液(0.1M，pH 7.0)洗涤5次，获得固定化的RFP。计算固定化RFP的固定量和固定化效率，相关计算公式如下：
RFP固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)-洗涤 液上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000014
固定化结果如表6所示。在pH为5-8的范围内固定化蛋白：环氧方法随着pH升高固定化的效率和活力回收率升高，固定化效率为31-41％，活力回收率为22-43％；本发明方法在pH为6-7时固定化效率和活力回收率最高，固定化效率为51-62％，活力回收率为51-67％。
表6 SpyCatcher-LX-1000EP在不同pH下固定SpyTag-RFP结果
Figure PCTCN2021099144-appb-000015
4.2不同温度下的固定化SpyTag-RFP
采用SpyCatcher-LX-1000EP在不同温度下固定化纯化的SpyTag-RFP。
本发明方法：称取50mg SpyCatcher-LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)分别在4℃、25℃、37℃下，25rpm转速下反应2h。
封闭对照：称取50mg经封闭反应之后的环氧载体，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)分别在4℃、25℃、37℃下，25rpm转速下反应2h。作为封闭对照组。
环氧方法：称取50mg LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)分别在4℃、25℃、37℃下，25rpm转速下反应2h。作为对照组。
同时取等量的SpyTag-RFP上清作为空白对照并检测其蛋白量。
反应完成后，通过离心分离出固定化RFP。用PBS缓冲液(0.1M，pH 7.0)洗涤5次，获得固定化的RFP。计算固定化RFP的固定量和固定化效率，相关计算公式如下：
RFP固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)-洗涤液上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000016
固定化结果如表7所示。在温度4℃、25℃和37℃下固定化蛋白：环氧方法随着温度升高，其固定化的效率和活力回收率升高，固定化效率为26-40％，活力回收率为34-47％；本发明方法对温度不敏感，固定化效率在4℃、25℃和37℃下均为62％，活力回收率为65-67％。
表7 SpyCatcher-LX-1000EP在不同温度下固定SpyTag-RFP
Figure PCTCN2021099144-appb-000017
4.3 SpyTag-RFP/SpyTag-GA的固定化
4.3.1固定化SpyTag-RFP
本发明方法：称取50mg SpyCatcher-LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。
环氧方法：称取50mg LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为对照组。
封闭对照：称取50mg经封闭反应之后的环氧载体，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化的SpyTag-RFP(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为封闭对照组。
同时取等量的SpyTag-RFP上清作为空白对照并检测其蛋白量。
反应完成后，通过离心分离出固定化RFP。用PBS缓冲液(0.1M，pH 7.0)洗涤5次，获得固定化的RFP。计算固定化RFP的固定量和固定化效率，相关计算公式如下：
RFP固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)-洗涤液上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000018
SDS-PAGE的分析结果如图5a所示。依照蛋白定量标准品，应用ImageJ凝胶定量分析软件对目的条带进行光密度分析，可计算得出融合蛋白SpyTag-RFP的固定化效率，结果如表8所示。封闭对照固定SpyTag-RFP的固定化效率为13.4％，环氧固定化方法的固定化效率为29.6％，本发明方法的固定效率为66.0％。这些荧光也可以从固定化载体上直接观测到，结果如图5b所示。
表8 SpyTag-RFP的固定化结果
Figure PCTCN2021099144-appb-000019
4.3.2从细胞裂解液中直接固定化SpyTag-RFP
本发明方法：称取50mg SpyCatcher-LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.2mL过表达SpyTag-RFP的大肠杆菌细胞裂解液上清，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。
环氧方法：称取50mg LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.2mL过表达SpyTag-RFP的大肠杆菌细胞裂解液上清，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为对照组。
封闭对照：称取50mg经封闭反应之后的环氧载体，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.2mL过表达SpyTag-RFP的大肠杆菌细胞裂解液上清，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为封闭对照组。
同时取等量的SpyTag-RFP上清作为空白对照并检测其蛋白量。
反应完成后，通过离心分离出固定化RFP。用PBS缓冲液(0.1M，pH 7.0)洗涤5次。获得固定化的SpyTag-RFP。计算固定化RFP的固定量、固定化效率及荧光回收率，相关计算公式如下：
RFP固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)-洗涤液上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000020
SDS-PAGE和荧光PAGE的分析结果分别如图6a和图6b所示。依照蛋白定量标准 品，应用ImageJ凝胶定量分析软件对目的条带进行光密度分析，可计算得出细胞裂解液中融合蛋白SpyTag-RFP的固定化效率，结果如表9所示。封闭对照固定SpyTag-RFP，其效率为42.3％，环氧固定化方法的固定化效率为62.7，本发明方法的固定效率提高到90.8％。SpyTag-RFP固定化的效果也可以直接通过固定化载体上的颜色观察到(图6c)。
表9 从细胞裂解液中固定化SpyTag-RFP的结果
Figure PCTCN2021099144-appb-000021
a.融合蛋白的投入量通过SDS-PAGE估算得到。
4.3.3固定化SpyTag-GA
本发明方法：称取50mg SpyCatcher-LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化后的SpyTag-GA(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。
环氧方法：称取50mg LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化后的SpyTag-GA(3.75mg/mL)，用微型旋转混匀仪(Gilson，Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为对照组。
封闭对照：称取50mg经封闭反应之后的环氧载体，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.75mg纯化后的SpyTag-GA(3.75mg/mL)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为封闭对照组。
同时取等量的SpyTag-GA上清作为空白对照并检测其蛋白量。
反应完成后，通过离心分离出固定化酶GA。用PBS缓冲液(0.1M，pH 7.0)洗涤5次。获得固定化的GA。计算固定化酶GA的固定量、固定化效率及酶活回收率，相关计算公式如下：
GA固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)-洗涤液上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000022
SDS-PAGE的分析结果如图7所示。依照蛋白定量标准品，应用ImageJ凝胶定量分析软件对目的条带进行光密度分析，可计算得出融合蛋白SpyTag-GA的固定化效率，结果如表10所示。封闭对照固定SpyTag-GA，其效率为28.4％，环氧固定化方法的固定化效率则为29.4％，而本发明方法的固定效率提高到91.5％。
表10 SpyTag-GA的固定化结果
Figure PCTCN2021099144-appb-000023
Figure PCTCN2021099144-appb-000024
4.3.4从细胞裂解液中直接固定化SpyTag-GA
称取50mg SpyCatcher-LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.2mL过表达SpyTag-GA的大肠杆菌细胞裂解液上清，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。
称取50mg LX-1000EP，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.2mL过表达SpyTag-GA的大肠杆菌细胞裂解液上清，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为对照组。
称取50mg经封闭反应之后的环氧载体，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，加入0.2mL过表达SpyTag-GA的大肠杆菌细胞裂解液上清，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应2h。作为封闭对照组。
同时取等量的SpyTag-GA上清作为空白对照并检测其蛋白量。
反应完成后，通过离心分离出固定化酶GA。用PBS缓冲液(0.1M，pH 7.0)洗涤5次。获得固定化的SpyTag-GA。计算SpyTag-GA的固定量、固定化效率及活力回收率，相关计算公式如下：
GA固定量(mg)＝固定前上清总蛋白量(mg)-固定后上清总蛋白量(mg)-洗涤液上清总蛋白量(mg)
Figure PCTCN2021099144-appb-000025
SDS-PAGE结果如图8所示。
依照蛋白定量标准品，应用ImageJ凝胶定量分析软件对目的条带进行光密度分析，可计算得出细胞裂解液中融合蛋白SpyTag-GA的固定化效率，结果如表11所示。封闭对照固定SpyTag-GA，其效率为28.5％，环氧方法的固定化效率则为25.0％，而本发明方法的固定效率提高到86.2％。
表11 细胞裂解液中SpyTag-GA的固定化结果
Figure PCTCN2021099144-appb-000026
a.融合蛋白的投入量通过SDS-PAGE估算得到。
4.4固定化SpyTag-RFP/GA活性检测
固定化SpyTag-RFP荧光检测：称量2mg(适量)固定化RFP，加入到黑色的平底 96孔板中，同时加入0.2mL PBS缓冲液(0.1M，pH 7.0)充分重悬，使用光栅型多功能酶标仪(TECAN,infinite M200PRO)在37℃下检测固定化SpyTag-RFP荧光强度，其激发波长为588nm，发射波长为635nm，增益值为100％。
固定化SpyTag-GA酶活力测定：称量4mg的固定化SpyTag-GA，加入0.5mL 0.1M PBS缓冲液(pH 7.0)，与0.5mL底物(在pH 7.0的0.1M PBS缓冲液中的1％(w/v)GL-7-ACA)混合，在37℃下孵育10分钟。然后取出0.2mL反应溶液，先加入0.7mL 20％(v/v)乙酸和0.05M NaOH的混合物(2：1，v/v)，然后加入0.1mL 0.5％(w/v)pDAB。将该混合物在25℃下再孵育10分钟，然后测量在415nm的吸光度。
固定化SpyTag-GA酶活力计算：
Figure PCTCN2021099144-appb-000027
ΔA/min—表示每分钟吸光度的变化值，即为斜率；
S—7-ACA的摩尔消光系数(1.576cm 2/μmoL)，通过标准曲线求得；
d—光程；(反应体系为0.2mL，光程约为0.5cm)；
Vt—表示反应液的总体积(1mL)；
M—表示固定化酶的称样量(0.004g)。
固定化SpyTag-RFP/SpyTag-GA活性检测结果如表12所示。封闭对照固定SpyTag-RFP和SpyTag-GA，其活性回收率分别为2.8％和1.1％，环氧方法的活性回收率分别为19.6％和2.7％，而本发明方法活性回收率分别提高到49.4％和86.4；此外，该固定化方法还可以从细胞裂解液中直接固定SpyTag-RFP/SpyTag-GA，环氧方法的活性回收率分别为36.3％和3.4％，本发明方法活性回收率分别提高到85.0％和91.2％。
表12 SpyTag-RFP/SpyTag-GA活性检测结果
Figure PCTCN2021099144-appb-000028
a：
Figure PCTCN2021099144-appb-000029
b：
Figure PCTCN2021099144-appb-000030
实施例5：比较不同环氧载体的固定化效果
5.1不同环氧载体分别固定SpyCatcher
用SpyCatcher修饰6种不同的环氧载体LX-1000EP(西安蓝晓科技新材料股份有限公司)、LX-107S(西安蓝晓科技新材料股份有限公司)、LX-103B(西安蓝晓科技新材 料股份有限公司)、HFA001(西安蓝晓科技新材料股份有限公司)、ECR8024(美国漂莱特有限公司)和ECR8285(美国漂莱特有限公司)，反应条件和固定化效率计算方法同实施例3。结果如表13所示。
6种树脂信息如下：
Figure PCTCN2021099144-appb-000031
表13 不同环氧载体的SpyCatcher固定化效率
Figure PCTCN2021099144-appb-000032
5.2 SpyCatcher修饰的不同环氧载体分别固定纯化的SpyTag-RFP
将SpyCatcher修饰的不同环氧载体分别固定化SpyTag-RFP，固定化条件、固定化效率和荧光回收率的计算方法同实施例4.3，结果如表14所示。
表14 SpyCatcher修饰的不同环氧载体固定化纯化的SpyTag-RFP结果
Figure PCTCN2021099144-appb-000033
Figure PCTCN2021099144-appb-000034
5.3 SpyCatcher修饰的环氧载体分别从细胞裂解液中固定SpyTag-GA
将SpyCatcher修饰的不同环氧载体分别从细胞裂解液中固定化SpyTag-GA，固定化条件、固定化效率和荧光回收率的计算方法同实施例4.3，结果如表15所示。
表15 SpyCatcher修饰的环氧载体从细胞裂解液中固定SpyTag-GA结果
Figure PCTCN2021099144-appb-000035
Figure PCTCN2021099144-appb-000036
5.4 SpyCatcher修饰的不同环氧载体直接从细胞裂解液固定SpyTag-RFP
将SpyCatcher修饰的不同环氧载体分别从细胞裂解液中固定化SpyTag-RFP，固定化条件、固定化效率和荧光回收率的计算方法同实施例4.3，结果如表16所示。
表16 SpyCatcher修饰的环氧载体从细胞裂解液中固定SpyTag-RFP结果
Figure PCTCN2021099144-appb-000037
Figure PCTCN2021099144-appb-000038
5.5固定化效率对比
5.5.1本发明方法与环氧方法固定化蛋白的固定化效率对比
把本发明方法和环氧方法，选择6种环氧载体和2种蛋白(包括纯化的蛋白SpyTag-RFP和含有目的蛋白SpyTag-RFP、SpyTag-GA的细胞裂解液)进行固定化，其固定化效率对比汇总如图9所示。本发明方法与环氧方法固定化效率统计数据如表17所示。本发明方法采用SpyCatcher修饰的改良树脂，固定化效率的平均值与中位值均高于环氧方法，而变异系数则远低于环氧方法，说明本发明方法的固定化效率比环氧方法更高、均一性更好。
表17 本发明方法与环氧方法固定化效率统计数据
Figure PCTCN2021099144-appb-000039
5.5.2不同环氧载体固定化效率对比
比较本发明方法SpyCatcher修饰树脂和环氧载体固定化蛋白质的固定化效率，结果如图10所示。对于大多数环氧载体，本发明方法的固定化效率显著高于或不低于环氧方法。
5.6活力回收率对比
5.6.1本发明方法与环氧方法固定化蛋白的活力回收率对比
把本发明方法和环氧方法，选择6种环氧载体和2种蛋白(包括纯化的蛋白SpyTag-RFP和含有目的蛋白SpyTag-RFP、SpyTag-GA的细胞裂解液)进行固定化，其活力回收率对比汇总如图11所示。本发明方法与环氧方法固定化效率统计数据如表18所示。本发明方法采用SpyCatcher修饰的改良树脂，活力回收率的平均值与中位值均高于环氧方法，而极差、四分位差和变异系数则远低于环氧方法，说明本发明方法的固定化效率比环氧方法更高、均一性更好。可见，现有技术方法固定化蛋白质效果不稳定，对蛋白种类和环氧载体型号比较敏感，对于某一种目的蛋白需要进行大量的环氧载体筛选工作。本发明方法对环氧载体进行改良，提高载体的均一性，减少对载体的筛选工作。
表18 本发明方法与环氧方法固定化效率统计数据
Figure PCTCN2021099144-appb-000040
Figure PCTCN2021099144-appb-000041
5.6.2不同环氧载体活力回收率对比
比较本发明方法SpyCatcher修饰载体和环氧载体固定化蛋白质的固定化效率，结果如图12所示。对于大多数环氧载体，本发明方法的活力回收率显著高于或不低于环氧方法。
5.7相对活力对比
5.7.1本发明方法与环氧方法固定化蛋白的相对活力对比
根据5.5和5.6的数据，根据公式：相对活力＝活力回收率/固定化效率×100％，计算相对活力，本发明方法和环氧方法固定化蛋白的相对活力对比汇总如图13所示。本发明方法与环氧方法固定化效率统计数据如表19所示。本发明方法采用SpyCatcher修饰的改良载体固定化的蛋白，其相对活力平均值与中位值分别为89.1％和96.9％，蛋白固定化后活力几乎没有损失，而环氧方法固定化的蛋白，其平均值与中位值分别为57.3％和48.4％，活力损失近一半。本发明方法固定化的相对活力变异系数远低于环氧方法，说明本发明方法的固定化效率比环氧方法更高、均一性更好。现有技术方法固定化蛋白质效果不稳定，对蛋白种类和环氧载体型号比较敏感，对于某一种目的蛋白需要进行大量的环氧载体筛选工作。本发明方法对环氧载体进行改良，提高载体的均一性，减少对载体的筛选工作。
表19 本发明方法与环氧方法固定化效率统计数据
Figure PCTCN2021099144-appb-000042
5.7.2不同环氧载体相对活力对比
比较本发明方法SpyCatcher修饰载体和环氧载体固定化蛋白质的相对活力，结果如图14所示。对于大多数环氧载体，本发明方法的相对活力显著高于或不低于环氧方法。
实施例6：比较不同批次的环氧载体采用本发明方法和现有方法的固定化效果
不同批次稳定性(固定化条件、固定化效率与活力回收率同实施例4.3)
Figure PCTCN2021099144-appb-000043
不同批次稳定性(固定化条件、固定化效率与活力回收率同实施例4.3)
Figure PCTCN2021099144-appb-000044
实施例7：SpyCatcher003修饰载体固定细胞裂解液中的SpyTag003-GA
本实施例中使用的寡聚核苷酸、引物如表20所示。
表20：寡聚核苷酸引物序列
Figure PCTCN2021099144-appb-000045
a引物下划线部分分别为限制性内切酶Xba I和Sac Ⅰ的识别位点。
7.1构建SpyCatcher003的表达质粒
本实施例中所构建的pET30a(+)-PTlinker-SpyCatcher003-His表达质粒(其中SpyCatcher003，SEQ ID NO：23)用于表达PTlinker-SpyCatcher003-His蛋白，构建方法同实施例1.1。
7.2构建SpyTag融合蛋白的表达质粒
构建质粒pET30a(+)-SpyTag003-Gslinker-GA-His(其中SpyTag003，SEQ ID NO：28)：假单胞菌SY-77(Pseudomonas sp.SY-77)的GA编码序列(SEQ ID NO：16)由生工生物科技有限公司(上海)经密码子优化后合成，GA的蛋白序列如SEQ ID NO：26所示。利用如表20中的寡聚核苷酸序列SpyTag003-GS-1、2、3和4进行PCR拼接、扩增获得Xba I-SpyTag003-Gslinker-Sac I多核苷酸片段，PCR拼接体系如表21所示。PCR反应使用Q5高保真DNA聚合酶，PCR扩增体系和程序如表22所示。
表21：寡聚核苷酸序列PCR拼接反应体系
Figure PCTCN2021099144-appb-000046
PCR拼接反应程序：a)98℃ 30sec；b)98℃ 10sec；c)62℃ 20sec；d)72℃ 10sec；e)步骤b)-d)，10循环；f)72℃ 2min；g)10℃ hold。
表22：PCR扩增反应体系
Figure PCTCN2021099144-appb-000047
PCR扩增反应程序：a)98℃ 30sec；b)98℃ 10sec；c)66℃ 20sec；d)72℃ 10sec；e)步骤b-d，30循环；f)72℃ 2min；g)4℃ hold。
反应结束后，对PCR扩增产物进行2％琼脂糖凝胶电泳检测，结果显示PCR扩增出与预期相符的目标DNA条带。使用超薄DNA产物纯化试剂盒纯化回收目标DNA，-20℃保存备用。
将纯化回收的目标DNA用限制性内切酶Xba Ⅰ和Sac Ⅰ(NEB，R0156L)进行双酶切后，与经同样酶双酶切的质粒pET30a(+)-SpyTag-Gslinker-GA-His进行连接，将连接产物转化到大肠杆菌BL21(DE3)感受态细胞，将转化细胞涂布于添加有50μg/mL卡那霉素(Kan)的LB平板上，选择阳性克隆，提取质粒，对其进行测序，测序结果表明所克隆的pET30a(+)-SpyTag003-Gslinker-GA-His序列正确。
7.3融合蛋白的表达、纯化和活性测定
将实施例7.1中构建的菌株(含有质粒pET30a(+)-PTlinker-SpyCatcher003-His)按1:50的接种量接种到含50μg/mL卡那霉素(Kan)的LB液体培养基中，过夜培养后， 转接到新鲜的LB(50μg/mL Kan)培养基中，并在37℃摇床中培养至对数期(OD 600＝0.4-0.6)，加入IPTG至终浓度为0.2mM，在30℃诱导6小时，测量菌浓度OD 600，收获细胞。
将实施例7.2中构建好的菌株(含有质粒pET30a(+)-SpyTag003-GSlinker-GA-His)接种到含50μg/mL Kan的LB液体培养基中，按1:50的接种量，将过夜培养的菌液转接到新鲜的LB(50μg/mL Kan)培养基中，并在37℃摇床中培养至对数期(OD 600＝0.4-0.6)，加入IPTG至终浓度为0.1mM，在18℃诱导12小时，测量菌浓度OD 600，收获细胞。
SDS-PAGE检测和定量、融合蛋白的纯化及透析和融合蛋白的酶活测定原理及方法同实施例2。
7.4 SpyCatcher003修饰载体的制备
制备方法及固定化效率计算同实施例3.1，结果如表23所示。当SpyCatcher003投入量为20mg/g载体时，固定化效率为90.1％。
表23 SpyCatcher003的固定化结果
Figure PCTCN2021099144-appb-000048
将得到的固定化SpyCatcher003-LX-1000EP加入12倍体积的封闭缓冲液(3M甘氨酸，pH 8.5)，用微型旋转混匀仪(Gilson,Roto-Mini Plus)在25℃、25rpm转速下反应24h。封闭完成后，离心去除封闭液，用PBS缓冲液(0.1M，pH 7.0)洗涤3次，存于4℃。
7.5从细胞裂解液中直接固定化SpyTag003-GA
固定方法、固定化效率计算同实施例4.3.4，结果如表24所示。封闭对照固定SpyTag003-GA，其效率为14.9％，环氧方法的固定化效率则为31.9％，本发明方法的固定效率为26.1％。
表24 细胞裂解液中SpyTag-GA的固定化结果
Figure PCTCN2021099144-appb-000049
a.融合蛋白的投入量通过SDS-PAGE估算得到。
7.6固定化SpyTag003-GA的活性检测
活性检测方法及酶活力计算同实施例4.4，结果如表25所示。环氧方法的活性回收率为33.1％，本发明方法活性回收率为23.8％。
表25 SpyTag003-GA活性检测结果
Figure PCTCN2021099144-appb-000050
a：
Figure PCTCN2021099144-appb-000051
本发明提供了一种基于SpyCatcher/SpyTag反应的酶固定化方法，具体实现该技术方案的方法和途径有很多，以上所述仅是本发明的示例性方案。对于本技术领域技术人员来说，在不脱离本发明的原理前提下，还可以做出若干改进和修饰，这些改进和修饰也应该视为在本发明的保护范围之内。本实施例中未明确的各组成部分均可用现有技术加以实现。

Claims (19)

  1. 一种SpyCatcher肽修饰的载体，其中所述载体，当未被SpyCatcher肽修饰时，包含能够与氨基(NH 2)反应的基团，其中所述SpyCatcher肽通过氨基与所述基团的反应而共价附着于所述载体，并且与所述载体连接的SpyCatcher肽能够与SpyTag肽形成异肽键，优选地，所述基团选自环氧基、醛基、酰亚胺、氰酸酯、亚胺碳酸酯和酰肼基。
  2. 权利要求1的SpyCatcher肽修饰的载体，其中所述SpyCatcher肽包含SEQ ID NO：21、22或23所示的氨基酸序列。
  3. 权利要求1或2的SpyCatcher肽修饰的载体，其中所述载体是环氧载体或氨基载体，优选地，所述载体由含有环氧基或氨基的聚丙烯酸酯制成。
  4. 一种SpyCatcher肽修饰的环氧树脂载体，其中所述SpyCatcher肽通过氨基与环氧基的反应而共价附着于所述载体，优选地，所述环氧树脂载体由聚丙烯酸酯制成。
  5. 权利要求4的SpyCatcher肽修饰的环氧树脂载体，其中所述环氧树脂的粒度为约100-1000μm，和/或所述环氧树脂的环氧基含量为约50-800μmol/g wet，优选地，所述环氧树脂的粒度为约100-350μm，和/或所述环氧树脂的环氧基含量为约50-100μmol/g wet。
  6. 权利要求1-5任一项的SpyCatcher肽修饰的载体，其与一种包含SpyTag肽和目的蛋白的融合蛋白连接，其中所述SpyCatcher肽与所述SpyTag肽形成异肽键，优选地，所述SpyTag肽包含选自SEQ ID NO：2、11-15和28任一所示的氨基酸序列。
  7. 权利要求6的SpyCatcher肽修饰的载体，其中所述目的蛋白是酶，优选选自戊二酰-7-氨基头孢烷酸酰基转移酶、葡萄糖异构酶、腈水合酶、青霉素酰胺酶、天冬氨酸酶、延胡索酸酶、氨基酰化酶、乳糖酶、天冬氨酸-β-脱羧酶和头孢菌素酰胺酶。
  8. 一种制备SpyCatcher肽修饰的载体的方法，包括：
    (1)提供包含能够与氨基(NH 2)反应的基团的载体，所述基团优选选自环氧基、醛基、酰亚胺、氰酸酯、亚胺碳酸酯和酰肼基；
    (2)在液体环境中将SpyCatcher肽与所述载体接触；任选地，在接触之前将所述基团活化，和/或在接触后，封闭所述载体；以及
    (3)任选地，分离SpyCatcher肽修饰的载体。
  9. 权利要求8的方法，其中所述SpyCatcher肽包含SEQ ID NO：21、22或23所示的氨基酸序列。
  10. 权利要求8或9的方法，其中所述载体是环氧载体或氨基载体，优选地，所述载体由聚丙烯酸酯制成。
  11. 一种固定化蛋白质的方法，包括：
    (1)提供权利要求1-5任一项所述的SpyCatcher肽修饰的载体或者根据权利要求8-10任一项的方法获得的SpyCatcher肽修饰的载体；
    (2)提供包含SpyTag肽和目的蛋白的融合蛋白，优选所述SpyTag肽包含SEQ ID NO：2、11-15和28任一所示的氨基酸序列；
    (3)将步骤(1)所述的载体与步骤(2)所述的融合蛋白在允许所述SpyCatcher肽与所述SpyTag肽形成异肽键的条件下相接触，任选地，在接触后，用洗涤缓冲液洗涤去除未固定化的融合蛋白；以及
    (4)任选地，分离与所述融合蛋白连接的载体。
  12. 权利要求11的方法，其中步骤(1)包括将SpyCatcher肽与未被SpyCatcher肽修饰的载体孵育，获得SpyCatcher肽修饰的载体；以及任选地，用封闭缓冲液处理SpyCatcher肽修饰的载体。
  13. 权利要求11或12的方法，其中所述目的蛋白是酶，优选选自戊二酰-7-氨基头孢烷酸酰基转移酶、葡萄糖异构酶、腈水合酶、青霉素酰胺酶、天冬氨酸酶、延胡索酸酶、氨基酰化酶、乳糖酶、天冬氨酸-β-脱羧酶和头孢菌素酰胺酶。
  14. 权利要求11-13任一项的方法，其中所述载体是环氧树脂或氨基载体，优选由聚丙烯酸酯制成。
  15. 试剂盒，其包含载体、SpyCatcher肽以及任选存在的SpyTag肽和目的蛋白例如酶的融合蛋白，其中所述载体和SpyCatcher肽单独存在或以权利要求1所述的SpyCatcher肽修饰的载体的形式存在。
  16. 权利要求15的试剂盒，其中所述SpyCatcher肽包含SEQ ID NO：21、22或23所示的氨基酸序列。
  17. 权利要求15或16的试剂盒，其中SpyTag肽优选包含选自SEQ ID NO：2、11-15和28任一所示的氨基酸序列。
  18. 权利要求15-17任一项的试剂盒，其中所述目的蛋白是酶，优选选自戊二酰-7-氨基头孢烷酸酰基转移酶、葡萄糖异构酶、腈水合酶、青霉素酰胺酶、天冬氨酸酶、延胡索酸酶、氨基酰化酶、乳糖酶、天冬氨酸-β-脱羧酶和头孢菌素酰胺酶。
  19. 权利要求15-18任一项的试剂盒，其中所述载体是环氧载体或氨基载体，优选由聚丙烯酸酯制成。
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