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

Abstract

The invention discloses an efficient polypeptide-polypeptide coupling system and method based on disordered protein coupling enzymes. By designing and splitting the protein domain CnaB2 and optimizing it, a three-component system is obtained: spy tag (SpyTag), BD tag (BDTag) and spy stapler enzyme (SpyStapler), so that SpyStapler can catalyze the coupling between SpyTag and BDTag. Coupling reaction, and prepare different fusion proteins containing SpyTag and BDTag two tags and carry out coupling or cyclization reaction. Based on the polypeptide-polypeptide coupling system of the present invention, an active protein with high purity and stable function can be obtained by means of gene coding; the reaction efficiency of the spy-stapling machine enzyme-coupled spy-tag and the BD-tag is high, and part of the spy is stapled after the reaction The organic enzyme falls off, and the coupling part of the obtained cyclization product has a smaller molecular weight. The protein tag of the present invention is suitable for a variety of functional proteins and can be fused and expressed at any position of the protein.

Description

A high-efficiency peptide-polypeptide coupling system and method based on disordered protein coupling enzyme

technical field

The present invention relates to the coupling technology of polypeptides, in particular to a polypeptide-polypeptide coupling system based on disordered protein coupling enzymes, and a method for coupling or realizing cyclization of proteins based on the system.

Background technique

Peptide/protein tags are widely used in protein purification, protein topology engineering, biological imaging and other fields. Protein tools such as SnoopLigase, Intein, Sortase, Butelase 1, and Asparagine Endopeptidase (OaAEP1) have been used for their high efficiency and specificity. It is considered to be a very effective biological polypeptide coupling method.

The emergence of "molecular superglue" technology provides a new class of protein labeling strategies. SpyTag and SpyCatcher systems, SnoopTag and SnoopCatcher systems and their mutants have demonstrated the specific affinity tagging properties of "molecular glue".

At present, there are few protein labeling tools based on protein structure splitting. Researchers split the protein domain CnaB2 to obtain SpyTag, KTag and SpyLigase, and split the protein domain RrgA A small probe tag (SnoopTagJr), a dog tag (DogTag) and a probe ligase (SnoopLigase) were obtained. These two systems require the addition of an additional small molecule additive N-trimethylamine oxide (TMAO) or glycerol, and the ligation efficiency of their ligases is not high. Currently, there is a particular need to develop efficient protein-based peptide-polypeptide coupling enzymes, especially those high-efficiency ligase systems that do not require additives.

SUMMARY OF THE INVENTION

The invention splits the protein domain CnaB2 to obtain a three-component system spy tag (SpyTag), BD tag (BDTag) and spy stapler enzyme (SpyStapler).

The following points should be paid attention to when conducting protein splitting: (1) The splitting site needs to be in a flexible chain or a relatively loose part of the protein domain; (2) The split polypeptide or protein should be re-identified and assembled. At present, there are mainly strategies based on structure splitting optimization and strategies based on computing simulation splitting optimization. There have been many reports and different applications for the method of protein resolution, such as the resolution of green fluorescent protein, the resolution of active protein and so on. The protein-protein or polypeptide-protein interaction based on protein-protein or polypeptide-protein interaction recombines and exerts corresponding functions. This strategy has been widely used in the field of bioimaging. However, there are fewer reports for splitting of protein tags. A series of biological coupling reaction pairs developed in recent years, such as spy tag (SpyTag), K tag (KTag) and spy ligase (SpyLigase), as well as splitting the protein domain RrgA to obtain a small probe tag (SnoopTagJr), dog tag ( DogTag) and probe ligase (SnoopLigase) and so on. Such tags are generally short peptides with a length of less than 20 amino acids, and the ligase part is a protein with a larger molecular weight of more than 20 amino acids. Such tags can be expressed in fusion with different proteins, cyclized active proteins or reacted to form active protein polymer chains. If a class of protein-chemical reaction pairs that can react efficiently under physiological conditions and have intracellular reactivity can be developed, then efficient intracellular cyclization can be achieved.

The invention performs protein splitting and optimization based on the protein structural domain CnaB2, and obtains a polypeptide-polypeptide coupling system of a three-component system, including a spy tag (SpyTag), a BD tag (BDTag) and a spy stapler enzyme (SpyStapler), The spy stapler enzyme (SpyStapler) can catalyze the coupling reaction between the spy tag (SpyTag) and the BD tag (BDTag), so that an efficient protein chain reaction can be performed. The fusion protein can realize the coupling or cyclization of intracellular or extracellular active proteins (such as dihydrofolate reductase).

Most preferably, the amino acid sequences of the spy tag (SpyTag), the BD tag (BDTag) and the spy stapler enzyme (SpyStapler) are respectively as SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No in the sequence listing: 3 shown. Among them, for the spy stapler enzyme (SpyStapler), the amino acid sequence corresponding to positions 52 to 111 of the spy catcher is its catalytic core, for example, glutamic acid (E) at position 31 in SEQ ID No: 3 is replaced For glutamine (Q), it loses the activity of catalyzing the formation of isopeptide bonds between SpyTag and BDTag, and outside the core region, the deletion, addition or substitution of a small number of amino acid residues (the substitution of amino acids with similar properties) has a negative effect on the catalytic function. Less affected.

The present invention also provides a protein coupling or cyclization method based on the polypeptide-polypeptide coupling system of the above-mentioned three-component system, the realization of which can occur intracellularly or extracellularly, including:

1) Construct the gene sequence of the fusion protein containing the tag (SpyTag and/or BDTag) and functional protein domain, and introduce it into the expression vector;

2) Introduce the expression vector constructed in step 1) into the cell, and express the corresponding fusion protein in the cell;

3) Simultaneously express the spy-stapler enzyme in the cell of step 2), so that the fusion protein undergoes a coupling or cyclization reaction in the cell; or, purify the fusion protein expressed in step 2), so that the fusion protein and the The spy stapler enzyme performs the extracellular reaction, and the spy stapler enzyme catalyzes the conjugation or cyclization of the fusion protein extracellularly.

One or more identical or different functional protein domains may be included in a protein building block. Tags can be located at the N-terminus, C-terminus and protein segments of the fusion protein. The fusion protein can be a disordered Elastin-like polypeptide (ELP) or an ordered fluorescent protein, dihydrofolate reductase, etc., including other functional proteins involved in the fields of medicine, agriculture, industry, and scientific research.

The following are some specific examples to illustrate the structure of building blocks containing protein tags:

(a) Elastin-SpyTag-Elastin (ELP-SpyTag-ELP): from the N-terminus to the C-terminus, the elastin-like protein, spy-tag, and elasto-like protein are respectively, and the corresponding gene sequences can be listed in the sequence table. SEQ ID No: 4, wherein amino acid residues 6-11 are 6×His, amino acid residues 16-93 are elastin, amino acid residues 96-105 are spy tags, and amino acid residues 108-189 The amino acid residues are elastin-like proteins.

(b) Elastin-BD Tag-Elastin (ELP-BDTag-ELP): from the N-terminus to the C-terminus, the elastin-like protein, BD tag, and elastin-like protein are respectively, and the corresponding gene sequences can be listed in the sequence table. SEQ ID No: 5, wherein amino acid residues 6-11 are 6×His, amino acid residues 16-93 are elastin, amino acid residues 96-120 are BD tags, and amino acid residues 121-201 The amino acid residues are elastin-like proteins.

(c) Spy tag-dihydrofolate reductase-BD tag (SpyTag-DHFR-BDTag): from the N-terminus to the C-terminus are spy tag, dihydrofolate reductase, BD tag, the corresponding gene sequence can be sequence SEQ ID No: 6 in the list, wherein amino acid residues 4-9 are 6×His, amino acid residues 22-34 are spy tags, amino acid residues 43-227 are dihydrofolate reductase, and amino acid residues 43-227 are dihydrofolate reductase. Amino acid residues 232-256 are BD tags.

In step 1), the genes of the spy tag and the BD tag are respectively or jointly constructed with the gene sequence of the functional protein domain to form the gene sequence of the fusion protein, preferably, the gene sequences of the spy tag and the BD tag are respectively in sequence SEQ ID No: 7 and SEQ ID No: 8 are shown in the list. Generally, the genes of spy tag and BD tag are constructed at both ends of the functional protein domain gene, and step 3) cyclization reaction occurs under the catalysis of spy stapler enzyme. If the genes of spy tag and BD tag are respectively constructed with the same or different functional protein domain genes, step 3) coupling between the same or different functional protein domains occurs under the catalysis of spy stapler enzyme linked reaction.

In step 3), the spy-stapling enzyme and the fusion protein are co-expressed intracellularly, or a 6×His tag is added to the N-terminal of the spy-stapling enzyme, and gene expression and purification are performed separately. Preferably, the gene of the spy stapler enzyme is shown in SEQ ID No: 9 in the sequence listing.

For the intracellular reaction product in the above step 3), if the fusion protein has a 6×His tag at the N-terminus, the broken extracellular solution can be purified using a Ni affinity chromatography column (such as Ni-NTA resin), Characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The protein expressed in the above step 3) is purified and subjected to extracellular reaction. The protein elution solution can be purified by fast flow chromatography, and spy-stapler enzyme reaction can be added under physiological conditions. Characterization by acrylamide gel electrophoresis.

In addition to elastin and dihydrofolate reductase, the method of the present invention is also applicable to various proteins such as fluorescent proteins.

The main advantages of the technology of the present invention are: the active protein with high purity and stable function can be obtained by means of gene coding; the reaction efficiency of the spy-stapler enzyme coupling spy-tag and the BD-tag is high, and part of the spy-stapler enzyme falls off after the reaction , the coupling part of the obtained cyclization product has a smaller molecular weight. The protein tag of the present invention is suitable for a variety of functional proteins and can be fused and expressed at any position of the protein.

Description of drawings

Figure 1 shows the in situ cyclization of SpyTag-DHFR-BDTag by SpyStapler (spy stapler enzyme)-mediated polypeptide-polypeptide (BDTag-SpyTag) coupling reaction in vivo, where: (a) SpyTag-DHFR-BDTag Schematic diagram of intracellular co-expression and cyclization with SpyStapler; (b) SDS-PAGE analysis of protein; (c) SEC analysis of protein; (d) mass spectrogram of dimer (DHFR) ₂ ; (e) ring Mass spectrum of the protein-like protein c-DHFR.

Figure 2 shows that the SpyStapler (spy stapler enzyme)-mediated peptide-polypeptide (BDTag-SpyTag) coupling reaction in vitro results in the coupling of ELP-SpyTag-ELP and ELP-BDTag-ELP to form a four-armed star compound, Among them: (a) schematic diagram of coupling reaction; (b) protein SDS-PAGE analysis result; (c) protein SEC analysis result; (d) mass spectrum of raw material reactant protein; (e) four-arm star ELP of reaction product mass spectrometry.

Figure 3 shows that SpyStapler (spy stapler enzyme)-mediated polypeptide-polypeptide (BDTag-SpyTag) coupling reaction in vitro results in the cyclization of SpyTag-DHFR-BDTag, where: (a) SpyTag-DHFR-BDTag extracellular Schematic diagram of the cyclization reaction; (b) protein SDS-PAGE analysis result; (c) protein SEC analysis result; (d) mass spectrum of raw reactant protein; (e) mass spectrum of reaction product.

Detailed ways

The present invention will be further described in detail through the following examples, but the scope of the present invention is not limited in any way.

Specific steps for preparing fusion protein containing protein tag: constructing a fusion protein containing 6×His tag (for protein purification), spy tag (SpyTag), BD tag (BDTag), elastin-like protein (ELP), dihydrofolate by recombinant genetic engineering technology The fusion protein of reductase (DHFR), namely the gene sequence of ELP-SpyTag-ELP, ELP-BDTag-ELP, SpyTag-DHFR-BDTag, was inserted into the expression vector, and the expression vector was transformed into E. coli for expression by molecular cloning technology. The target fusion protein is obtained by a series of protein purification methods such as affinity chromatography. The fusion proteins include ELP-SpyTag-ELP, ELP-BDTag-ELP, SpyTag-DHFR-BDTag.

Using 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 performance liquid Liquid chromatography (LC-MS) and other methods were used to characterize the molecular weight, topology and other properties of fusion proteins and their reaction products.

Example 1: Transformation and expression of fusion protein target gene

The plasmids of pQE-80 L ELP-SpyTag-ELP and pQE-80 L ELP-BDTag-ELP were constructed, confirmed by sequencing, transferred into BL21(DE3) competent cells and plated, and cultured in an incubator overnight at 37 ^o C . Pick out monoclonal colonies on the plate into LB medium containing 0.10 mg/mL ampicillin sodium and incubate overnight at 37 ^o C in a shaking incubator. Take the medium from the overnight culture and add it to 1 L LB medium containing 0.10 mg/mL ampicillin sodium at a ratio of 1:100, shake it at 37 ^o C and culture it to an OD ₆₀₀ of 0.4-0.6, add isopropyl-β-D - Thiogalactopyranoside (IPTG) to a final concentration of 1 mM to induce protein expression in E. coli. Change the temperature of the shaking incubator to 30 ^o C.

The plasmid pACYCDuet-1 SpyTag-DHFR-BDTag (MCS1)-SpyStapler (MCS2) was constructed, confirmed by sequencing, transferred into BL21 (DE3) competent cells and plated, and cultured in an incubator overnight at 37 ^o C. Pick out monoclonal colonies on the plate into LB medium containing 0.50 mg/mL chloramphenicol, and cultivate overnight at 37 ^o C in a shaking incubator. Take the medium from the overnight culture and add it to 1 L LB medium containing 0.50 mg/mL chloramphenicol at a ratio of 1:100, shake it at 37 ^o C and culture it to an OD ₆₀₀ of 0.4-0.6, add isopropyl-β-D - Thiogalactopyranoside (IPTG) to a final concentration of 1 mM to induce protein expression in E. coli. The shaking incubator temperature was changed to 16 ^o C for overnight incubation.

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. For protein spy-staplerase (SpyStapler), spy-staplerase mutant (SpyStapler-EQ) and fusion protein SpyTag-DHFR-BDTag purified by native conditions, use native buffer A (20 mM sodium dihydrogen phosphate) , 300 mM NaCl, 10 mM imidazole, pH = 8.0). The resuspension was sonicated for 20 minutes in a 4 ^o C ice-water bath (5 sec work, 5 sec interval, 40% intensity). The disrupted solution was centrifuged at 20,000 g for 20 minutes in a high-speed floor-standing centrifuge at 4 ^o C, and the lysed supernatant was retained. The supernatant was mixed with the equilibrated affinity resin Ni-NTA resin, and the mixture was uniformly mixed by rotating at 4 ^o C for 1 hour. Pour the mixture into the empty column, wait for the resin to settle evenly, and discard the lysate. Rinse the protein of interest with native buffer B (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, pH = 8.0) followed by native buffer C (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride) , 250 mM imidazole, pH = 8.0) to elute the target protein. The collected target protein is concentrated using an ultrafiltration tube, and the obtained concentrate is purified by a protein rapid purification system at 4 ^o C.

For proteins ELP-SpyTag-ELP, ELP-BDTag-ELP purified with denaturing conditions, use denaturing buffer A (20 mM sodium dihydrogen phosphate, 300 mM NaCl, 10 mM imidazole, 8 M urea, pH = 8.0) Resuspended. The resuspension was sonicated for 20 minutes in a 4 ^o C ice-water bath (5 sec work, 5 sec interval, 40% intensity). The disrupted solution was centrifuged at 20,000 g for 20 minutes in a high-speed floor-standing centrifuge at 4 ^o C, and the lysed supernatant was retained. The supernatant was mixed with the equilibrated affinity resin Ni-NTA resin, and the mixture was uniformly mixed by rotating at 4 ^o C for 1 hour. Pour the mixture into the empty column, wait for the resin to settle evenly, and discard the lysate. Rinse the protein of interest with natural buffer B (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, 8 M urea, pH = 8.0), followed by natural buffer C (50 mM sodium dihydrogen phosphate, 300 mM urea). mM NaCl, 250 mM imidazole, 8 M urea, pH = 4.0) to elute the protein of interest. The collected target protein is concentrated using an ultrafiltration tube, and the obtained concentrate is purified by a protein rapid purification system at 4 ^o C.

Example 3: Characterization of fusion proteins

1 mL of the protein eluate was purified on a gel permeation column Superdex 200 Increase 10/300 GL on an AKTA protein purification system (AKTA Avant, GE Healthcare). The mobile phase was PBS and the flow rate was 0.5 mL/min. Target peaks were collected by monitoring A ₂₈₀ .

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 (ABSCIEX) . The results are shown in Figure 1. In vivo SpyStapler-mediated polypeptide-polypeptide (BDTag-SpyTag) coupling reaction resulted in in situ cyclization of SpyTag-DHFR-BDTag.

Example 4: Intracellular responses and characterization of fusion proteins

According to the purification results of the protein rapid purification system, take 20 μL of the 8 mL outflow volume and 12 mL outflow volume sample, add 5× loading buffer and place it in the gene amplification instrument to boil to stop the reaction. Add 20 μL of protein elution solution to 5× loading buffer and place it in the gene amplification instrument to boil. In the control group, SpyTag-DHFR-BDTag with a final concentration of linearity was taken and added with 5× loading buffer and placed in a gene amplification apparatus to boil. The protein was characterized by polyacrylamide gel electrophoresis, and the reactants and products of the protein were quantified by a multifunctional fluorescence analyzer. The results are shown in Figure 1. When SpyTag-DHFR-BDTag and SpyStapler were co-expressed intracellularly, a polypeptide-polypeptide (BDTag-SpyTag) coupling reaction mediated by SpyStapler (spy stapler enzyme) resulted in in situ cyclization of SpyTag-DHFR-BDTag.

Example 5: Extracellular responses and characterization of fusion proteins

The concentrations of ELP-SpyTag-ELP, ELP-BDTag-ELP, SpyStapler, SpyStapler-EQ were measured with an ultra-micro spectrophotometer (P330, Implen). Take ELP-SpyTag-ELP and ELP-BDTag-ELP to the final protein concentration of 10 μM in the system, and the final concentration of SpyStapler or SpyStapler-EQ is 30 μM. Add 1×PBS (pH=7.4) to a volume of 20 μL, place in a gene amplification apparatus and incubate at 4 ^o C for 8 hours. For the experimental group containing N-trimethylamine oxide (TMAO), the final concentration of TMAO was 1.5 M. After reaching the reaction time, 5× loading buffer was added and placed in a gene amplification instrument to boil to stop the reaction. The protein was characterized by polyacrylamide gel electrophoresis, and the reactants and products of the protein were quantified by a multifunctional fluorescence analyzer. The results are shown in Figure 2. The SpyStapler (spy stapler enzyme)-mediated polypeptide-polypeptide (BDTag-SpyTag) coupling reaction resulted in the coupling of ELP-SpyTag-ELP and ELP-BDTag-ELP to form a four-armed star. type compound.

Example 6: Extracellular responses and characterization of fusion proteins

The concentrations of SpyTag-DHFR-BDTag and SpyStapler were measured with an ultra-micro spectrophotometer (P330, Implen). Take SpyTag-DHFR-BDTag to the final concentration of 10 μM in the system, and the final concentration of SpyStapler to 20 μM. Add 1×PBS (pH=7.4) to a volume of 20 μL, place in a gene amplification apparatus and incubate at 4 ^o C for 8 hours. After reaching the reaction time, add 5× sample buffer and place it in the gene amplification instrument to boil to stop the reaction. The protein was characterized by polyacrylamide gel electrophoresis, and the reactants and products of the protein were quantified by a multifunctional fluorescence analyzer. As a result, as shown in Fig. 3, the cyclized and dimerized dihydrofolate reductase was obtained by the extracellular cyclization of active protein dihydrofolate reductase mediated by SpyStapler (spy stapler enzyme).

SEQUENCE LISTING

<110> Peking University

<120> A high-efficiency peptide-polypeptide coupling system and method based on disordered protein coupling enzyme

<130> WX2019-03-166

<150> CN2018112219357

<151> 2018-10-19

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Claims (9)

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.

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