CN111073925A - High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme - Google Patents

High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme Download PDF

Info

Publication number
CN111073925A
CN111073925A CN201910995483.6A CN201910995483A CN111073925A CN 111073925 A CN111073925 A CN 111073925A CN 201910995483 A CN201910995483 A CN 201910995483A CN 111073925 A CN111073925 A CN 111073925A
Authority
CN
China
Prior art keywords
protein
spy
polypeptide
enzyme
tag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910995483.6A
Other languages
Chinese (zh)
Other versions
CN111073925B (en
Inventor
张文彬
吴夏泠
刘雅杰
刘栋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University
Original Assignee
Peking University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University filed Critical Peking University
Publication of CN111073925A publication Critical patent/CN111073925A/en
Application granted granted Critical
Publication of CN111073925B publication Critical patent/CN111073925B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • C12N9/003—Dihydrofolate reductase [DHFR] (1.5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/01003—Dihydrofolate reductase (1.5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a high-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme. A three-component system is obtained by designing and splitting a protein structural domain CnaB2 and optimizing the protein structural domain CnaB 2: spyware tag (SpyTag), BD tag (BDTag) and spy stitcher enzyme (spystpler) such that spystpler can catalyze the coupling reaction between SpyTag and BDTag, and prepare different fusion proteins containing both SpyTag and BDTag and perform the coupling or cyclization reaction. Based on the polypeptide-polypeptide coupling system, the active protein with high purity and stable function can be obtained by utilizing a gene coding mode; the spy stapler enzyme coupling spy label and BD label reaction efficiency is high, part of spy stapler enzyme falls off after reaction, and the molecular weight of the coupling part of the obtained cyclization product is small. The protein tag of the invention is suitable for various functional proteins and can be fused and expressed at any position of the protein.

Description

High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme
Technical Field
The invention relates to a polypeptide coupling technology, in particular to a polypeptide-polypeptide coupling system based on disordered protein coupling enzyme and a method for coupling or realizing cyclization of proteins based on the system.
Background
Polypeptide/protein tags are widely used in the fields of protein purification, protein topology engineering, biological imaging, and the like. Protein tools such as ligase (snoopLigase), Intein (Intein), Sortase (Sortase), sphenophagase (butterfly 1) and asparaginyl endopeptidase (OaAEP1) are considered to be very effective biological polypeptide coupling methods due to their high efficiency and specificity.
The advent of the "molecular superglue" technology provides a new class of protein labeling strategies. The spy tag (SpyTag) and spy catcher (SpyCatcher) systems, the SnoopTag (SnoopTag) and snooper (snoopcaptor) systems and their mutants have demonstrated the specific affinity labelling properties of "molecular glues".
At present, few protein labeling tools are available based on protein structure resolution, researchers resolve the protein domain CnaB2 to obtain spy tags (SpyTag), K tags (KTag) and spy ligase (spy ligase), and resolve the protein domain RrgA to obtain small probe tags (snoeptagjr), dog tags (DogTag) and probe ligase (snoipligase). The two systems need to add additional small-molecule additives of trimethylamine N-oxide (TMAO) or glycerol, and the ligation efficiency of the ligase is not high. There is a particular need to develop highly efficient protein-based polypeptide-polypeptide conjugate enzymes, particularly those highly efficient ligase systems that do not require additives.
Disclosure of Invention
The present invention is based on the protein domain CnaB2 to obtain a three-component system spy tag (SpyTag), BD tag (BDTag) and spy stitcher enzyme (SpyStapler).
When protein resolution is carried out, the following points need to be paid attention: (1) the split site requires a flexible chain or relatively loose portion in the protein domain; (2) and re-identifying and assembling the split polypeptide or protein. At present, strategies based on structural splitting optimization and strategies based on computational simulation splitting optimization are mainly available. There have been many reports on the methods for protein resolution and various applications, such as resolution of green fluorescent protein, resolution of active protein, and the like. The protein after being resolved is recombined based on protein-protein or polypeptide-protein interaction and plays corresponding functions. This strategy has been widely used in the field of biological imaging. However, less reports have been made of resolution of protein tags. A series of bioconjugate reaction pairs developed in recent years, such as spy tag (SpyTag), K tag (KTag) and spy ligase (spy ligase), and the splitting of the protein domain RrgA to give small probe tag (snooptag jr), dog tag (DogTag) and probe ligase (SnoopLigase), among others. Such tags are typically short peptides within twenty amino acids in length, and the ligase portion is a protein of larger molecular weight of more than twenty amino acids. Such tags can be expressed as fusions with different proteins, cyclizing the active protein or reacting to form active macromolecular chains of the protein. If a protein chemical reaction pair which can react efficiently under physiological conditions and has intracellular reactivity could be developed, efficient intracellular cyclization reaction could be achieved.
The invention is based on protein domain CnaB2 to carry out protein resolution and optimization, and obtains a polypeptide-polypeptide coupling system of a three-component system, which comprises a spy tag (spyTag), a BD tag (BDTag) and spy stitcher enzyme (spyStapler), so that the spy stitcher enzyme (SpyStapler) can catalyze the coupling reaction between the spy tag (spyTag) and the BD tag (BDTag), thereby carrying out efficient in-chain reaction, and coupling or cyclization of intracellular or extracellular active proteins (such as dihydrofolate reductase) can be realized by preparing different fusion proteins containing the spyTag and the BDTag.
Most preferably, the amino acid sequences of the spy tag (SpyTag), the BD tag (BDTag) and the spy stitcher enzyme (SpyStapler) are respectively as shown in SEQ ID nos: 1. SEQ ID No: 2 and SEQ ID No: 3, respectively. Among them, spyware enzymes (spystaplers) have an amino acid sequence corresponding to positions 52 to 111 of spyware as a catalytic core, and for example, SEQ ID No: the substitution of glutamine (Q) for glutamic acid (E) at position 31 in 3 deprives the activity of catalyzing the formation of isopeptide bonds by SpyTag and BDTag, while the deletion, addition or substitution of a small number of amino acid residues (substitution of amino acids of similar nature) has less influence on the catalytic function outside the core region.
The invention also provides a protein coupling or cyclization method based on the polypeptide-polypeptide coupling system of the three-component system, which can be realized in cells and can also be realized outside the cells, and comprises the following steps:
1) constructing a gene sequence of a fusion protein containing the tag (SpyTag and/or BDTag) and a functional protein structural domain, and introducing the gene sequence into an expression vector;
2) introducing the expression vector constructed in the step 1) into cells, and expressing corresponding fusion proteins in the cells;
3) simultaneously expressing the spy stitcher enzyme in the cells in the step 2) to enable the fusion protein to generate coupling or cyclization reaction in the cells; or purifying the fusion protein expressed in the step 2), and carrying out extracellular reaction between the fusion protein and the spy stitcher enzyme, wherein the spy stitcher enzyme catalyzes coupling or cyclization reaction of the fusion protein in the extracellular space.
The protein building blocks may include one or more identical or different functional protein domains. The tag may be located at the N-terminus, C-terminus, and in the protein segment of the fusion protein. The fusion protein can be disordered Elastin-like protein (ELP) or ordered fluorescent protein, dihydrofolate reductase and the like, and comprises other functional proteins related in the fields of medicine, agriculture, industry and scientific research.
The structure of the protein tag-containing building block is illustrated below by some specific examples:
(a) elastin-spy tag-elastin (ELP-SpyTag-ELP): from the N end to the C end, the gene sequences are respectively elastin, spy tag and elastin, and the corresponding gene sequences can be SEQ ID No: 4, wherein the 6 th to 11 th amino acid residues are 6 XHis, the 16 th to 93 th amino acid residues are elastin, the 96 th to 105 th amino acid residues are spy tags, and the 108 th and 189 th amino acid residues are elastin.
(b) elastin-BD tag-elastin (ELP-BDTag-ELP): from N terminal to C terminal, it is elastic protein, BD label, elastic protein, and its corresponding gene sequence can be SEQ ID No: 5, wherein the 6 th-11 th amino acid residues are 6 XHis, the 16 th-93 th amino acid residues are elastin, the 96 th-120 th amino acid residues are BD labels, and the 121 st-201 th amino acid residues are elastin.
(c) Spy tag-dihydrofolate reductase-BD tag (SpyTag-DHFR-BDTag): from the N end to the C end, spy tags, dihydrofolate reductase tags and BD tags are respectively arranged, and the corresponding gene sequences can be SEQ ID No: 6, wherein the amino acid residues at the 4 th to the 9 th positions are 6 XHis, the amino acid residues at the 22 th to the 34 th positions are spy tags, the amino acid residues at the 43 th to the 227 th positions are dihydrofolate reductase, and the amino acid residues at the 232 rd and the 256 th positions are BD tags.
In the step 1), genes of the spy tag and the BD tag and gene sequences of a functional protein structural domain are constructed into gene sequences of fusion proteins respectively or together, preferably, the gene sequences of the spy tag and the BD tag are respectively shown as SEQ ID No: 7 and SEQ ID No: shown in fig. 8. In general, genes of the spy tag and the BD tag are constructed at both ends of a functional protein domain gene, and step 3) is performed by a cyclization reaction catalyzed by spy stitcher enzyme. If genes of the spy label and the BD label are respectively constructed together with the same or different functional protein domain genes, step 3) is that the coupling reaction between the same or different functional protein domains occurs under the catalytic action of spy stitcher enzyme.
In step 3), the spy stitase and the fusion protein are co-expressed intracellularly, or a 6 × His tag is added to the N-terminal of the spy stitase, and gene expression and purification are performed separately. Preferably, the spy stapler enzyme gene is shown as SEQ ID No: shown at 9.
For the intracellular reaction product in the above step 3), if the fusion protein has a 6 × His tag at the N-terminus, the disrupted extracellular solution can be purified using a Ni affinity chromatography column (e.g., Ni-NTA resin) and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Purifying the expressed protein in the step 3) and carrying out extracellular reaction, purifying the protein elution solution by using a fast flow chromatography, adding spy stapler enzyme under physiological conditions for reaction, and characterizing by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The method of the present invention is applicable to various proteins such as fluorescent proteins in addition to elastin and dihydrofolate reductase.
The main technical advantages of the invention are as follows: the active protein with high purity and stable function can be obtained by utilizing a gene coding mode; the spy stapler enzyme coupling spy label and BD label reaction efficiency is high, part of spy stapler enzyme falls off after reaction, and the molecular weight of the coupling part of the obtained cyclization product is small. The protein tag of the invention is suitable for various functional proteins and can be fused and expressed at any position of the protein.
Figure BDA0002239590880000041
Drawings
Figure 1 shows that in vivo polypeptide-polypeptide (BDTag-SpyTag) conjugation mediated by SpyStapler (spy stitcher) results in situ cyclization of SpyTag-DHFR-BDTag, wherein: (a) schematic representation of intracellular co-expression and cyclization of SpyTag-DHFR-BDTag and SpyStapler; (b) SDS-PAGE analysis of the protein; (c) SEC analysis results of the protein; (d) dimer (DHFR)2Mass spectrogram of (1); (e) mass spectrum of the cyclic protein c-DHFR.
FIG. 2 shows that in vitro SpyStapler (spy stitcher) mediated polypeptide-polypeptide (BDTag-SpyTag) conjugation results in the conjugation of ELP-SpyTag-ELP and ELP-BDTag-ELP to form a four-arm star-shaped compound, wherein: (a) schematic representation of the coupling reaction; (b) protein SDS-PAGE analysis results; (c) protein SEC analysis results; (d) mass spectrometry of the raw reactant protein; (e) mass Spectrometry of the reaction product four-arm star-shaped ELP.
Fig. 3 shows that in vitro SpyStapler (spy stitcher) mediated polypeptide-polypeptide (BDTag-SpyTag) conjugation results in cyclization of SpyTag-DHFR-BDTag, wherein: (a) schematic representation of the extracellular cyclization reaction of SpyTag-DHFR-BDTag; (b) protein SDS-PAGE analysis results; (c) protein SEC analysis results; (d) mass spectrometry of the raw reactant protein; (e) mass spectrum of the reaction product.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
The preparation of the fusion protein containing the protein tag comprises the following specific steps: constructing fusion protein containing 6 XHis tag (for protein purification), spy tag (SpyTag), BD tag (BDtag), Elastin (ELP) and dihydrofolate reductase (DHFR) by recombinant genetic engineering technology, namely gene sequences of ELP-SpyTag-ELP, ELP-BDtag-ELP and SpyTag-DHFR-BDtag, inserting the gene sequences into an expression vector, transforming the expression vector into escherichia coli by molecular cloning technology for expression, and obtaining the target fusion protein by a series of protein purification methods such as affinity chromatography. The fusion protein comprises ELP-SpyTag-ELP, ELP-BDTag-ELP and SpyTag-DHFR-BDTag.
The properties of the fusion protein and the reaction product thereof, such as molecular weight, topological structure and the like, are characterized by methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Size Exclusion Chromatography (SEC), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), liquid chromatography-ultra high performance liquid chromatography (LC-MS) and the like.
Example 1: transformation and expression of fusion protein target gene
Plasmids of pQE-80L ELP-SpyTag-ELP and pQE-80L ELP-BDTag-ELP are constructed, transferred into BL21(DE3) competent cells after sequencing confirmation and plated, and are put in an incubator overnightCulturing at 37 ℃. Single colonies were picked on the plates and cultured overnight in LB medium containing 0.10mg/mL ampicillin sodium at 37 ℃ in a shaking incubator. Adding overnight culture medium into 1L LB medium containing 0.10mg/mL ampicillin sodium at a ratio of 1: 100, shake-culturing at 37 deg.C to OD600Adding isopropyl- β -D-thiogalactopyranoside (IPTG) to a final concentration of 1mM to induce the expression of the protein in Escherichia coli, wherein the temperature of the shaking incubator is changed to 30 ℃.
Plasmid pACYCDuet-1 SpyTag-DHFR-BDTag (MCS1) -SpyStapler (MCS2) was constructed, transferred into BL21(DE3) competent cells after sequencing confirmation, plated, and cultured in an incubator overnight at 37 ℃. Single colonies were picked on the plates into LB medium containing 0.50mg/mL chloramphenicol and incubated overnight at 37 ℃ in a shaking incubator. Adding overnight culture medium into 1L LB medium containing 0.50mg/mL chloramphenicol at a ratio of 1: 100, shake-culturing at 37 deg.C to OD6000.4-0.6, adding isopropyl- β -D-thiogalactopyranoside (IPTG) to a final concentration of 1mM to induce the expression of the protein in E.coli, and culturing overnight at 16 ℃ instead of the temperature of the shaking incubator.
Example 2: purification of fusion proteins
After the expression, the cells were collected by high-speed centrifugation at 4000g for 20 minutes, and the supernatant was discarded. The proteins spywater (spystpler), spywater mutant (spystpler-EQ) and fusion protein SpyTag-DHFR-BDTag purified in natural conditions were resuspended in non-denaturing buffer a (20mM sodium dihydrogen phosphate, 300mM sodium chloride, 10mM imidazole, pH 8.0). The resuspension was sonicated for 20 minutes in an ice-water bath at 4 ℃ (5 seconds on, 5 seconds apart, 40% strength). The disruption solution was centrifuged at 20000g for 20 minutes at 4 ℃ using a high speed floor centrifuge, and the lysis supernatant was retained. Mixing the supernatant with well-balanced affinity resin Ni-NTA resin, and uniformly mixing at 4 deg.C for 1 hr. Pouring the mixed solution into an empty column, waiting for the resin to uniformly settle, and discarding the lysate. The target protein was washed with natural buffer B (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 20mM imidazole, pH 8.0), and then eluted with natural buffer C (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 250mM imidazole, pH 8.0). The collected target protein is concentrated by using an ultrafiltration tube, and the obtained concentrated solution is purified by a protein rapid purification system at the temperature of 4 ℃.
The proteins ELP-SpyTag-ELP and ELP-BDTag-ELP purified under denaturing conditions were resuspended in denaturing buffer A (20mM sodium dihydrogen phosphate, 300mM sodium chloride, 10mM imidazole, 8M urea, pH 8.0). The resuspension was sonicated for 20 minutes in an ice-water bath at 4 ℃ (5 seconds on, 5 seconds apart, 40% strength). The disruption solution was centrifuged at 20000g for 20 minutes at 4 ℃ using a high speed floor centrifuge, and the lysis supernatant was retained. Mixing the supernatant with well-balanced affinity resin Ni-NTA resin, and uniformly mixing at 4 deg.C for 1 hr. Pouring the mixed solution into an empty column, waiting for the resin to uniformly settle, and discarding the lysate. The target protein was washed with natural buffer B (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 20mM imidazole, 8M urea, pH 8.0), and then eluted with natural buffer C (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 250mM imidazole, 8M urea, pH 4.0). The collected target protein is concentrated by using an ultrafiltration tube, and the obtained concentrated solution is purified by a protein rapid purification system at the temperature of 4 ℃.
Example 3: characterization of fusion proteins
1mL of protein eluate was purified by passing through gel permeation column Superdex 200 Increate 10/300GL on AKTA protein purification system (AKTAAvant, GE Healthcare). The mobile phase was PBS and the flow rate was 0.5 mL/min. By monitoring A280The target peak was collected.
The molecular weight of the purified product was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) or high performance liquid chromatography-electrospray mass spectrometry (LC-MS) using a 5800MALDI-TOF/TOF analyzer (AB SCIEX). As shown in fig. 1, in vivo polypeptide-polypeptide (BDTag-SpyTag) conjugation mediated by SpyStapler (spy stitcher) resulted in situ cyclization of SpyTag-DHFR-BDTag.
Example 4: intracellular response and characterization of fusion proteins
According to the purification result of the protein rapid purification system, 20. mu.L of each of 8mL and 12mL samples was taken, 5 Xloading buffer was added and the mixture was boiled in a gene amplification apparatus, and the reaction was stopped. mu.L of the protein elution solution was added to 5 Xloading buffer and placed in a gene amplification apparatus and boiled. The control group was taken at a final linear concentration of SpyTag-DHFR-BDTag and added to 5 Xloading buffer and placed in a gene amplifier to boil. Proteins were characterized by polyacrylamide gel electrophoresis and protein reagents and products were quantified using a multifunctional fluorescence analyzer, the results of which are shown in FIG. 1. When SpyTag-DHFR-BDTag and spystpler are co-expressed intracellularly, the polypeptide-polypeptide (BDTag-SpyTag) conjugation reaction mediated by spystpler (spy stitcher) results in situ cyclization of SpyTag-DHFR-BDTag.
Example 5: extracellular reaction and characterization of fusion proteins
The concentrations of ELP-SpyTag-ELP, ELP-BDTag-ELP, SpyStapler and SpyStapler-EQ were measured with a ultramicro spectrophotometer (P330, Implen). The ELP-SpyTag-ELP and the ELP-BDTag-ELP are respectively taken to ensure that the final concentration of protein in the system is 10 mu M and the final concentration of SpyStapler or SpyStapler-EQ is 30 mu M. 1 XPBS (pH 7.4) was added to a volume of 20. mu.L and incubated in a gene amplifier at 4 ℃ for 8 hours. For the group containing trimethylamine N-oxide (TMAO), TMAO was added at a final concentration of 1.5M, and after the reaction time, 5 Xloading buffer was added and the mixture was boiled in a gene amplification apparatus to stop the reaction. The proteins were characterized by polyacrylamide gel electrophoresis and the reactants and products of the proteins were quantified using a multifunctional fluorescence analyzer. As a result, as shown in FIG. 2, the polypeptide-polypeptide (BDTag-SpyTag) coupling reaction mediated by SpyStapler (spy stitcher) resulted in the coupling of ELP-SpyTag-ELP and ELP-BDTag-ELP to form a four-arm star-shaped compound.
Example 6: extracellular reaction and characterization of fusion proteins
The concentrations of SpyTag-DHFR-BDTag and SpyStapler were measured by an ultramicrospectrophotometer (P330, Implen). SpyTag-DHFR-BDTag is taken to be the system with the final concentration of 10 mu M and the SpyStapler with the final concentration of 20 mu M. 1 XPBS (pH 7.4) was added to a volume of 20. mu.L and incubated in a gene amplifier at 4 ℃ for 8 hours. After the reaction time is reached, 5 Xloading buffer is added and the reaction is stopped by boiling the sample in a gene amplification instrument. The proteins were characterized by polyacrylamide gel electrophoresis and the reactants and products of the proteins were quantified using a multifunctional fluorescence analyzer. As a result, as shown in FIG. 3, cyclized and dimerized dihydrofolate reductase was obtained by the active protein dihydrofolate reductase extracellular cyclization reaction mediated by SpyStapler (spy stitcher enzyme).
SEQUENCE LISTING
<110> Beijing university
<120> high-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme
<130>WX2019-03-166
<150>CN2018112219357
<151>2018-10-19
<160>9
<170>PatentIn version 3.5
<210>1
<211>13
<212>PRT
<213> Artificial sequence
<400>1
Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys
1 5 10
<210>2
<211>25
<212>PRT
<213> Artificial sequence
<400>2
Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu
1 5 10 15
Ala Gly Ala Thr Met Glu Leu Arg Asp
20 25
<210>3
<211>64
<212>PRT
<213> Artificial sequence
<400>3
Val Glu Ala Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln
1 5 10 15
Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr
20 25 30
Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val
35 40 45
Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly Gly
50 55 60
<210>4
<211>205
<212>PRT
<213> Artificial sequence
<400>4
Met Lys Gly Ser Ser His His His His His His Val Asp Gly His Gly
1 5 10 15
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu
20 25 30
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
35 40 45
Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val
50 55 60
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
65 70 75 80
Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Leu Ala
85 90 95
His Ile Val Met Val Asp Ala Tyr Lys Pro ThrLys Thr Ser Val Pro
100 105 110
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly
115 120 125
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
130 135 140
Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
145 150 155 160
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val
165 170 175
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu Leu Asp
180 185 190
Gly Pro Gln Gly Ile Trp Gly Gln Leu Glu Lys Lys Met
195 200 205
<210>5
<211>217
<212>PRT
<213> Artificial sequence
<400>5
Met Lys Gly Ser Ser His His His His His His Val Asp Gly His Gly
1 5 10 15
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu
20 25 30
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
35 40 45
Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val
50 55 60
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
65 70 75 80
Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Leu Ala
85 90 95
Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala
100 105 110
Gly Ala Thr Met Glu Leu Arg Asp Thr Ser Val Pro Gly Val Gly Val
115 120 125
Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro
130 135 140
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
145 150 155 160
Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
165 170 175
Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly
180 185 190
Val Pro Gly Val Gly Val Pro Gly Gly Leu Leu Asp Gly Pro Gln Gly
195 200 205
Ile Trp Gly Gln Leu Glu Lys Lys Met
210 215
<210>6
<211>256
<212>PRT
<213> Artificial sequence
<400>6
Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg
1 5 10 15
Gly Ser His Met Gly Ala His Ile Val Met Val Asp Ala Tyr Lys Pro
20 25 30
Thr Lys Gly Ser Gly Gly Ser Gly Met Ile Ser Leu Ile Ala Ala Leu
35 40 45
Ala Val Asp Arg Val Ile Gly Met Glu Asn Ala Met Pro Trp Asn Leu
50 55 60
Pro Ala Asp Leu Ala Trp Phe Lys Arg Asn Thr Leu Asn Lys Pro Val
65 70 75 80
Ile Met Gly Arg His Thr Trp Glu Ser Ile Gly Arg Pro Leu Pro Gly
85 90 95
Arg Lys Asn Ile Ile Leu Ser Ser Gln Pro Gly Thr Asp Asp Arg Val
100 105 110
Thr Trp Val Lys Ser Val Asp Glu Ala Ile Ala Ala Cys Gly Asp Val
115 120 125
Pro Glu Ile Met Val Ile Gly Gly Gly Arg Val Tyr Glu Gln Phe Leu
130 135 140
Pro Lys Ala Gln Lys Leu Tyr Leu Thr His Ile Asp Ala Glu Val Glu
145 150 155 160
Gly Asp Thr His Phe Pro Asp Tyr Glu Pro Asp Asp Trp Glu Ser Val
165 170 175
Phe Ser Glu Phe His Asp Ala Asp Ala Gln Asn Ser His Ser Tyr Cys
180 185 190
Phe Glu Ile Leu Glu Arg Arg Gly Ser Gly Gly Ser Gly Gly Ala Met
195 200 205
Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp
210 215 220
Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg
225 230 235 240
Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp
245 250 255
<210>7
<211>39
<212>DNA
<213> Artificial sequence
<400>7
gcccacatcg tgatggtgga cgcctacaag ccgacgaag 39
<210>8
<211>75
<212>DNA
<213> Artificial sequence
<400>8
gctacccata ttaaattctc aaaacgtgat gaggacggca aagagttagc tggtgcaact 60
atggagttgc gtgat 75
<210>9
<211>192
<212>DNA
<213> Artificial sequence
<400>9
gtcgaggcta gcggtaaaac tattagtaca tggatttcag atggacaagt gaaagatttc 60
tacctgtatc caggaaaata tacatttgtc gaaaccgcag caccagacgg ttatgaggta 120
gcaactgcta ttacctttac agttaatgag caaggtcagg ttactgtaaa tggcaaagca 180
actaaaggtg gc 192

Claims (10)

1. A polypeptide-polypeptide coupling system is obtained by performing protein resolution and optimization based on a protein domain CnaB2 and comprises a spy label, a BD label and spy stitcher enzyme, wherein the spy stitcher enzyme can catalyze the coupling reaction between the spy label and the BD label.
2. The polypeptide-polypeptide conjugate system of claim 1, wherein the spy tag, BD tag and spy stitcher enzyme have amino acid sequences as set forth in SEQ ID nos: 1. SEQ ID No: 2 and SEQ ID No: 3, respectively.
3. A method of protein coupling or cyclization based on the polypeptide-polypeptide coupling system of claim 1 or 2 comprising:
1) constructing a gene sequence of a fusion protein containing the spy tag, the BD tag and the functional protein domain as defined in claim 1 or 2 and introducing the gene sequence into an expression vector;
2) introducing the expression vector into cells, and expressing the fusion protein corresponding to the constructed gene in the cells;
3) simultaneously expressing the spy stitcher enzyme of claim 1 or 2 in a cell to cause a coupling or cyclization reaction of the fusion protein in the cell; or purifying the expressed fusion protein, so that the spy stitcher enzyme catalyzes the coupling or cyclization reaction of the fusion protein in the extracellular space.
4. The method according to claim 3, wherein the functional protein domains in step 1) comprise one or more functional protein domains, which may be the same or different, and wherein the spy tag and the BD tag are located at the N-terminus, C-terminus and/or in a protein segment of the fusion protein.
5. The method according to claim 4, characterized in that in step 1) the genes of the spy and BD-tags are constructed separately or together with the genes of the functional protein domain.
6. The method of claim 3, wherein the functional protein domain in step 1) is selected from one or more of an elastin-like protein, a fluorescent protein, and a dihydrofolate reductase.
7. The method of claim 3, wherein the fusion protein has a 6 × His tag at the N-terminus and is purified using a Ni affinity chromatography column.
8. The method of claim 3, wherein the spy label and BD label have the gene sequences shown in SEQ ID No: 7 and SEQ ID No: shown in fig. 8.
9. The method as claimed in claim 3, wherein in step 3), the spyware enzyme is co-expressed intracellularly with the fusion protein, or the spyware enzyme is separately expressed and purified with the addition of a 6 × His tag to the N-terminus of the spyware enzyme.
10. The method of claim 3, wherein the spy stitcher enzyme gene is as set forth in SEQ ID NO: shown at 9.
CN201910995483.6A 2018-10-19 2019-10-18 High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme Active CN111073925B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2018112219357 2018-10-19
CN201811221935 2018-10-19

Publications (2)

Publication Number Publication Date
CN111073925A true CN111073925A (en) 2020-04-28
CN111073925B CN111073925B (en) 2022-04-26

Family

ID=70310587

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910995483.6A Active CN111073925B (en) 2018-10-19 2019-10-18 High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme

Country Status (1)

Country Link
CN (1) CN111073925B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021233330A1 (en) * 2020-05-21 2021-11-25 北京大学 Method for biosynthesis of protein heterogeneous catenane

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105061581A (en) * 2015-09-17 2015-11-18 北京大学 Preparation method for genetically coded holoprotein catenane
CN106591345A (en) * 2016-12-26 2017-04-26 华侨大学 Method for separation and purification and immobilization integration of recombinant double enzyme
CN108026148A (en) * 2015-06-05 2018-05-11 牛津大学创新有限公司 Fusion protein synthetic method and product

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108026148A (en) * 2015-06-05 2018-05-11 牛津大学创新有限公司 Fusion protein synthetic method and product
CN105061581A (en) * 2015-09-17 2015-11-18 北京大学 Preparation method for genetically coded holoprotein catenane
CN106591345A (en) * 2016-12-26 2017-04-26 华侨大学 Method for separation and purification and immobilization integration of recombinant double enzyme

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
WEN-BIN ZHANG等: "《Controlling macromolecular topology with genetically encoded SpyTag-SpyCatcher chemistry》", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 *
XIA-LING WU等: "《An intrinsically disordered peptide-peptide stapler for highly efficient protein ligation both in vivo and in vitro》", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 *
方晶等: "《可基因编码的多肽-蛋白质化学反应对》", 《高分子学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021233330A1 (en) * 2020-05-21 2021-11-25 北京大学 Method for biosynthesis of protein heterogeneous catenane

Also Published As

Publication number Publication date
CN111073925B (en) 2022-04-26

Similar Documents

Publication Publication Date Title
US11161899B2 (en) Method using split inteins to generate protein conjugates
CN108026148B (en) Method and product for synthesis of fusion protein
JP2017060491A (en) Aldehyde tag and use thereof in site-specific protein modification
CN113195521B (en) Mtu delta I-CM intein variants and uses thereof
Korepanova et al. Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein
Zhao et al. A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus
EP3750911A1 (en) Cysteine-free inteins
CN111073925B (en) High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme
Kofoed et al. Semisynthesis of an active enzyme by quantitative click ligation
Greimann et al. Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae: cloning, expression, purification, and activity assays
CN111705050A (en) Preparation method and application of novel halophilic archaea extracellular protease
US7879578B2 (en) Self-assembled proteins and related methods and protein structures
Chilakapati et al. Characterization and Expression Profiling of Recombinant Parathyroid Hormone (rhPTH) Analog 1–34 in Escherichia coli, Precise with Enhanced Biological Activity
Palei et al. Preparation of Semisynthetic Peptide Macrocycles Using Split Inteins
Lee et al. Salt-sensitive intein for large-scale polypeptide production
CN112851765B (en) Method for covalently linking protein or peptide to nucleic acid
US20240182530A1 (en) Polypeptides that interact with peptide tags at loops or termini and uses thereof
CN114685679A (en) Spyware mutant, preparation method thereof and application thereof in fluorescent protein system
Tang Improving the use of asparaginyl endopeptidase for biocatalytic applications
CN117264081A (en) Dipeptide receptor agonist and preparation method and application thereof
WO2018025826A1 (en) Antibody fused with labeling protein
KR20210079235A (en) Method for enhancing soluble expression of target proteins by using fusion protein of whep domain
CN102021193B (en) Method for producing polypeptide by combining biological expression of protein with chemical cracking technique
WO2022263559A1 (en) Production of cross-reactive material 197 fusion proteins
Ponomarenko et al. Production in bacterial cell a recombinant analog of proinsulin with the proper disulfide bonds

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant