CN111073925B

CN111073925B - High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme

Info

Publication number: CN111073925B
Application number: CN201910995483.6A
Authority: CN
Inventors: 张文彬; 吴夏泠; 刘雅杰; 刘栋
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2018-10-19
Filing date: 2019-10-18
Publication date: 2022-04-26
Anticipated expiration: 2039-10-18
Also published as: CN111073925A

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 (OaAEP 1) 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-like-spy tag-elastin-like (ELP-SpyTag-ELP): from the N end to the C end, the gene sequences of the elastin-like protein, the spy tag and the elastin-like protein are respectively shown as 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 the elastin-like protein, the 96 th to 105 th amino acid residues are the spy tag, and the 108 th and 189 th amino acid residues are the elastin-like protein.

(b) Elastin-like protein-BD tag-elastin-like protein (ELP-BDTag-ELP): from the N end to the C end, the target gene is respectively an elastin-like protein, a BD label and an elastin-like protein, and the corresponding gene sequences can be SEQ ID No: 5, wherein the 6 th to 11 th amino acid residues are 6 XHis, the 16 th to 93 th amino acid residues are the elastin-like protein, the 96 th to 120 th amino acid residues are the BD labels, and the 121 st and 201 th amino acid residues are the elastin-like protein.

(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-like proteins 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.

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) a schematic 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) a schematic 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-like protein (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 for expression by using molecular cloning technology, and obtaining 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 overnight at 37^oAnd C, culturing. Single colonies were picked on plates to contain 0.10 mg/mL ampicillin sodium in LB medium, in a shaking incubator 37^oC culture overnight. The overnight culture medium was added to 1L LB medium containing 0.10 mg/mL ampicillin sodium at a ratio of 1: 100, and the mixture was incubated at 37^oC shake culture to OD₆₀₀0.4-0.6, isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce E.coli expressed protein. The temperature of the shaking incubator is changed to 30^oAnd C, culturing.

The plasmid of pACYCDuet-1 SpyTag-DHFR-BDTag (MCS1) -SpyStapler (MCS2) is constructed, transferred into BL21(DE3) competent cells after sequencing confirmation, plated, and cultured in an incubator overnight at 37^oAnd C, culturing. Single colonies were picked on plates into LB medium containing 0.50 mg/mL chloramphenicol in a shake incubator 37^oC culture overnight. The overnight culture medium was added to 1L LB medium containing 0.50 mg/mL chloramphenicol at a ratio of 1: 100, 37^oC shake culture to OD₆₀₀0.4-0.6, isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce E.coli expressed protein. The temperature of the shaking incubator is changed to 16^oC overnight culture.

Example 2: purification of fusion proteins

After the expression, the cells were collected by high-speed centrifugation at 4000 g 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 (20 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, pH = 8.0). Resuspension in 4^oC sonication in an ice-water bath for 20 minutes (5 seconds work, 5 seconds apart, 40% strength). At 4^oUnder C, the disruption solution was centrifuged at 20000 g for 20 minutes using a high speed floor centrifuge, and the lysis supernatant was retained. Mixing the supernatant with well-balanced affinity resin Ni-NTA resin at 4^oC, rotating and uniformly mixing for 1 hour. Pouring the mixed solution into an empty column, waiting for the resin to uniformly settle, and discarding the lysate. The reaction mixture was washed with natural buffer B (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole,pH = 8.0) and then eluted with natural buffer C (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH = 8.0). Collecting the target protein and concentrating with ultrafiltration tube to obtain concentrated solution 4^oAnd C, purifying by a protein rapid purification system.

For the proteins ELP-SpyTag-ELP, ELP-BDTag-ELP purified with denaturing conditions, they were resuspended in denaturing buffer A (20 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, 8M urea, pH = 8.0). Resuspension in 4^oC sonication in an ice-water bath for 20 minutes (5 seconds work, 5 seconds apart, 40% strength). At 4^oUnder C, the disruption solution was centrifuged at 20000 g for 20 minutes using a high speed floor centrifuge, and the lysis supernatant was retained. Mixing the supernatant with well-balanced affinity resin Ni-NTA resin at 4^oC, rotating and uniformly mixing for 1 hour. Pouring the mixed solution into an empty column, waiting for the resin to uniformly settle, and discarding the lysate. The protein of interest was washed with natural buffer B (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, 8M urea, pH = 8.0) and then eluted with natural buffer C (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, 8M urea, pH = 4.0). Collecting the target protein and concentrating with ultrafiltration tube to obtain concentrated solution 4^oAnd C, purifying by a protein rapid purification system.

Example 3: characterization of fusion proteins

1 mL of protein eluate was purified by passing through gel permeation column Superdex 200 Increate 10/300 GL on AKTA protein purification System (AKTA Avant, 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 5800 MALDI-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 8 mL and 12 mL 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 placed in a gene amplification apparatus 4^oC culture 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). Taking SpyTag-DHFR-BDTag to the final concentration of the system10 μ M, final SpyStapler concentration 20 μ M. 1 XPBS (pH = 7.4) was added to a volume of 20. mu.L and placed in a gene amplification apparatus 4^oC culture 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

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Claims

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, and the amino acid sequences of the spy label, the BD label and the spy stitcher enzyme are respectively shown as SEQ ID No: 1. SEQ ID No: 2 and SEQ ID No: 3, respectively.

2. A method of protein coupling or cyclization based on the polypeptide-polypeptide coupling system of claim 1 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, 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 spywstitase of claim 1 in cells to allow the fusion protein to undergo a coupling or cyclization reaction intracellularly; 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.

3. The method according to claim 2, wherein the fusion protein in step 1) comprises 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.

4. The method according to claim 3, 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.

5. The method of claim 2, 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.

6. The method of claim 2, wherein the fusion protein has a 6 × His tag at the N-terminus and is purified using a Ni affinity chromatography column.

7. The method of claim 2, wherein the spy label and BD label gene sequences are as shown in SEQ ID nos: 7 and SEQ ID No: shown in fig. 8.

8. The method as claimed in claim 2, 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.

9. The method of claim 2, wherein the spy stitcher enzyme gene is as set forth in SEQ ID No: shown at 9.