CN110272913B - Protein coupling method based on soyabean - Google Patents
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Abstract
The invention discloses a protein coupling method based on soyabean alkylation. According to the invention, the secondary components (SpyTag, BDTag and SpyStapler) in the SpyTag-SpyCatcher compound are re-connected, an artificially designed chain entanglement structure is introduced, a protein active template method combining entanglement and catalysis is developed, and mechanical bond coupling of different target proteins connected in a catenane form can be realized. The method is used for carrying out heterogeneous solonization on disordered protein or folded protein, and the structure and the performance of the protein are not obviously influenced. The soxhlet alkylation process can also realize the preparation of direct expression in cells through a co-expression system. Since the catenane structure has conformational restriction and steric control on the target protein, it can improve the stability of the target protein. The invention realizes the concise synthesis of protein heterosoxohydrocarbons, and is a novel protein coupling strategy with great value.
Description
Technical Field
The invention relates to preparation of biological macromolecular protein, in particular to coupling of protein through heterogeneous solvolysis, and application of solvolysis to improvement of stability of target protein.
Background
While conventional protein coupling relies primarily on covalent bond coupling to obtain a linear or branched backbone fusion structure, examples of protein coupling using mechanical bonds are rare and less considered for the spatial distribution of protein domains in the conjugate. The cyclic structure may improve the stability of the protein due to its restriction on the conformation of the protein backbone. Currently, work in the literature is directed primarily to circularizing individual proteins. The cyclization of multiple proteins greatly weakens the domain-limiting effect, so that the improvement of the protein stability is limited. The soxhlet hydrocarbon is a topological structure obtained by mechanical interlocking of two or more cyclic structures, wherein the target cyclic proteins are connected by mechanical bonds, and the spatial distribution of the target proteins can be regulated by regulating the relative positions of the target proteins in the soxhlet hydrocarbon structure, so the soxhlet hydrocarbon is a novel protein coupling strategy which is worthy of being researched.
Currently, few reports are made about the preparation of artificial protein catenanes. The earliest example was cyclization of the entangled dimerization motif of the tumor suppressor p53 using native chemical ligation to give the catenated structure of p53 entangled dimer (Yan, L.Z. and Dawson, P.E., Angew.chem.int.Ed.2001,113, 3737-3739.; Blankenship, J.W., and Dawson, P.E., Protein Sci.2007,16, 1249-. However, this method is based on polypeptide synthesis and is hardly suitable for the production of high molecular weight proteins. The Zhang group utilizes p53 entanglement dimer to combine with SpyTag-SpyCatcher reaction pair to realize the catenanization of disordered protein ELP and folded protein in order intracellularly, and finds that the catenanization can improve the enzymolysis resistance, the thermal stability and the enzyme catalytic activity of target protein (Liu, D, et al, ACS.Cent.Sci.2017,3,473-481, Wang, X.W.and Zhang, W.B., Angew.Chem.int.Ed.2016,55, 3442-3446; Wang, X.W.and Zhang, W.B., Angew.Chem.Ed.2017, 56, 13985-13989.). All the work obtains homogeneous protein catenane, and no reports on artificial protein heterogeneous catenane exist at present.
Active template synthesis is an effective strategy for preparing small molecule topological structure developed in more than ten years. The catalyst not only serves as a reaction template to pre-assemble each reaction component, but also serves as a catalytic center to promote covalent bond coupling among the reaction components, so that a mechanical bond is formed in situ, and a target topological structure comprising rotaxane and catehydrocarbon is obtained. The method combines assembly and catalysis, and is a powerful means for constructing topological molecules. However, this strategy has not been applied to macromolecular systems.
Disclosure of Invention
The invention designs an active template strategy suitable for preparing protein heterogeneous catenane, develops a plurality of protein active template systems, and realizes the preparation of a plurality of protein heterogeneous catenane structures, thereby providing a novel protein coupling method. The coupling method has no influence on the structure and activity of the target protein, and improves the enzymolysis resistance, heat-resistant induced unfolding resistance and freeze-thaw denaturation resistance of the target protein to a certain extent.
The preparation of protein heterosoxohydrocarbons requires the following three conditions to be met: (1) chain entanglement structures are present to achieve mechanical interlocking; (2) a site-directed chemical coupling reaction is present to achieve cyclization of the components; (3) the two components of the catenane are different. The reported protein catenane structures are all based on the tangled dimer structure of p53 and subsequent cyclization strategies (such as native chemical ligation or polypeptide-protein reaction pair), and therefore, most of the protein catenane structures are homogeneous, and the application of the protein catenane structures in protein coupling is greatly limited. The invention develops a new method for preparing a protein heterogeneous catenane structure: active template method. Spyware tag (SpyTag) and spyware (SpyCatcher) are a pair of polypeptide-protein reactions derived from streptococcus pyogenes, where SpyTag is a short peptide of only thirteen amino acids and SpyCatcher is a protein with a molecular weight of 13 kDa. Studies have shown that SpyCatcher can be further split into a northern tag (BDtag) and spy stitcher enzyme (SpyStapler), where BDtag contains a reactive lysine and SpyStapler contains a catalytic site, glutamic acid. SpyStapler catalyzes the formation of a covalent bond between SpyTag and BDTag. Aiming AT the reconnection among the secondary components (SpyTag, BDTag and SpyStapler), the invention introduces an artificially designed chain entanglement structure into a SpyTag-Spycatcher compound, and further develops an active reaction template system (abbreviated as AT-Spy) based on SpyTag/BDTag/SpyStapler. The method comprises the steps of firstly utilizing a separation type intein coupling strategy to cyclize SpyStapler and a target protein 1, then utilizing recombination of BDtag-target protein 2-SpyTag and the former to realize the winding structure of the two components, further realizing the winding of the latter under the action of SpyStapler, and further obtaining the protein heterocatene structure in one step. The method is a highly modular method, wherein the target protein can also be various disordered proteins and folded proteins, and the coupling does not affect the structure and activity of the folded protein. The catenane structure has significantly improved thermal stability compared to its open chain control (SpyTag-BDTag/SpyStapler complex), on the one hand it can compensate for the loss of activity due to the introduction of flexible chains at both ends of the active protein, and on the other hand it can also confer better resistance to enzymatic hydrolysis, resistance to thermally induced unfolding and resistance to mechanical denaturation to the target protein.
The research of the invention comprises the following aspects:
A. the active template for preparing the protein catenane structure is designed, the active template consists of BDTag, SpyTag and SpyStapler, and the artificially designed chain entanglement can be realized by adjusting the connection sequence of the BDTag, the SpyTag and the SpyStapler, so that the catenane structure is prepared.
B. Starting from an active template AT-Spy, target proteins (such as disordered proteins [ elastin like ELP ], folded proteins dihydrofolate reductase [ DHFR ] and fluorescent proteins [ GFPrm ]) are introduced to realize the soxhlet alkylation of different types of target proteins.
C. Basic structural characterization of the protein catenane structure.
D. Testing the properties of the protein catenane structure.
E. The direct cell expression preparation of protein heterogeneous catenane is realized by utilizing a co-expression system.
F. And optimizing the active template to prepare the active template with higher reaction efficiency.
According to the above research, the present invention provides the following technical solutions:
a method for soxhlation-based protein coupling comprising the steps of:
1) designing two precursor sequences of protein catenane reaction, namely a circular fusion protein sequence of SpyStapler-target protein 1 and a linear fusion protein sequence of BDtag-target protein 2-Spytag;
2) constructing coding genes corresponding to the two precursor sequences in the step 1), and introducing a vector plasmid;
3) expressing the gene constructed in the step 2) by using cells;
4) purifying the expressed protein to obtain the heterogeneous catenane structures of the target protein 1 and the target protein 2.
In the step 1), typical amino acid sequences of BDTag, SpyTag and SpyStapler are respectively as shown in SEQ ID NOs: 3. SEQ ID NO: 1 and SEQ ID NO: 4, or a mutant thereof which retains coupling reactivity. The mutant is a peptide chain derived by substituting, deleting or adding 1 or more amino acid residues on the basis of the amino acid sequences of BDTag, SpyTag and SpyStapler, and the substituted, deleted or added amino acid residues do not influence the coupling reaction functions of the BDTag, the SpyTag and the SpyStapler, namely do not influence lysine (K at position 12 in SEQ ID NO: 3), aspartic acid (D at position 7 in SEQ ID NO: 1) and catalytic site glutamic acid (E at position 28 in SEQ ID NO: 4) which participate in isopeptide bond formation. Substitution, deletion or addition of amino acid residues, and detection of related functions can be accomplished by conventional techniques in the art. SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: 13 the amino acid sequences of the mutants BDTag002, SpyTag002 and SpyStapler002 are given, respectively.
In the step 1), in order to realize the cyclization of the SpyStapler-target protein 1 fusion protein, the C-terminal part and the N-terminal part of the separated intein can be fused at the N-terminal and the C-terminal respectively during design, and after the gene sequence is translated, the two separated intein structures are recombined and then react and are self-sheared, so that the cyclic C-SpyStapler-target protein 1 is obtained; the cyclization process may also utilize other cyclization or conjugation strategies such as enzyme-mediated cyclization, protein reaction pair-mediated conjugation, native chemical ligation, and the like.
Further, for purification, step 1) was to design a His tag sequence at the N-terminus of the fusion protein, and protein purification was performed by nickel affinity chromatography at step 4).
The step 2) may be to construct the encoding genes corresponding to the fusion protein sequence of the circular spystpler-target protein 1 and the fusion protein sequence of the linear BDTag-target protein 2-SpyTag on expression vectors respectively, or to construct the two on the same co-expression vector.
One option is that step 3) expresses two reaction precursors (a fusion protein of a cyclic spystpler-target protein 1 and a fusion protein of a linear BDTag-target protein 2-SpyTag) in different cells, respectively, then the two reaction precursors are purified in step 4), and the two reaction precursors are mixed in vitro under a certain condition, in the process, the two reaction precursors are subjected to structural recombination, and then isopeptide bonds are formed in situ, so that the preparation of the heterogeneous catenane structure is realized.
Alternatively, the two reaction precursors are co-expressed in the cells in the step 3), the two reaction precursors are subjected to structural recombination in the cells, so that isopeptide bonds are formed in situ, the preparation of the heterogeneous catenane structure is realized, and the protein with the heterogeneous catenane structure is purified in the step 4).
In the present invention, the active template consisting of BDTag, SpyTag and SpyStapler is a three-component reaction system obtained by resolving the SpyTag-SpyCatcher protein reaction pair again, which respectively comprises lysine (K), aspartic acid (D) and catalytic site glutamic acid (E) for participating in isopeptide bond formation. In the observation of the crystal structure of the SpyTag-SpyCatcher complex, the isomer of the catenane structure of the SpyTag-SpyCatcher complex can be obtained when the three components BDTag, SpyStapler and SpyTag are re-connected. This was resolved to give two precursors, linear BDTag-SpyTag (abbreviated as BD-A) and cyclic SpyStapler (c-SpyStapler, abbreviated as c-S). The cyclic SpyStapler can be prepared by trans-cleavage of the isolated intein. When the two reaction precursors (BD-A and c-S) are mixed and recombined under certain conditions, isopeptide bonds can be formed in situ, and the preparation of the heterosoxhlet hydrocarbon structure is realized.
The amino acid sequences of the SpyTag and the SpyCatcher are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The amino acid sequences of BDTag, SpyTag and SpyStapler are respectively shown as SEQ ID NO: 3. SEQ ID NO: 1 and SEQ ID NO: 4, respectively.
On the basis of the active template consisting of BDTag, SpyTag and SpyStapler, other proteins, such as disordered proteins or folded proteins, can be introduced into the connection region and used for realizing the heterogeneous solonization of target proteins. For example, the cyclic SpyStapler is introduced with the elastin-like protein ELP1 to prepare c-SpyStapler-ELP1 (abbreviated as c-S-E1), and simultaneously, BDTag and SpyTag are fused at two ends of another section of elastin-like protein ELP2 with different lengths to prepare BDTag-ELP2-SpyTag (abbreviated as BD-E2-A), so that the catenation of the elastin-like protein ELP is realized. Or folding proteins (such as DHFR and GFPrm) are introduced to respectively prepare reaction precursors BDTag-DHFR-SpyTag (abbreviated as BD-DHFR-A) and c-SpyStapler-GFPrm (abbreviated as c-S-GFPrm), and the two different folding proteins are subjected to heterogeneous solonation to realize the coupling of the two different folding proteins.
The structure of the reaction precursor in the preparation of the protein catenane structure is illustrated below by specific examples:
(a) BDTag-ELP 2-SpyTag: from the N end to the C end, the protein is respectively a 6 XHis tag, a reaction motif BDtag, a target protein ELP2 and a reaction motif SpyTag, wherein the connecting part of the BDtag and the ELP2 is inserted with the enzyme cutting site of TVMV enzyme. The corresponding amino acid sequence of BDTag-ELP2-SpyTag is SEQ ID NO: 5, wherein the 6-11 amino acid sequence is a 6 XHis tag, the 14-42 amino acid sequence is BDTag, the 51-57 amino acid residues are TVMV enzyme recognition sites, the 60-141 amino acid sequence is a target protein ELP2, and the 142-154 amino acid sequence is SpyTag.
(b) c-SpyStapler-ELP 1: the original translated amino acid sequence is SEQ ID NO: 6, from the N end to the C end, an intein C-terminal part, a target protein ELP1 ', a reaction motif SpyStapler, a target protein ELP1 ' and a separated intein N-terminal part are respectively arranged, wherein the amino acid sequence at the 5-43 position is the separated intein C-terminal part, the amino acid sequence at the 46-51 position is a 6 XHis tag, the amino acid sequence at the 54-78 position is a first section of ELP (ELP1 '), the amino acid sequence at the 81-141 position is the reaction motif SpyStapler, the amino acid sequence at the 144- 233 position is a second section of the target protein ELP (ELP1), the amino acid residue at the 236-242 position is a TEV enzyme digestion site, and the amino acid sequence at the 243-344 position is the separated intein N-terminal part. After the gene sequence is translated, the two parts of separated inteins react and are self-sheared, so that the cyclic c-SpyStapler-ELP1 is obtained.
(c) BDTag-DHFR-SpyTag: from the N end to the C end, the protein is respectively 6 × His tag, reaction motif BDTag, target protein DHFR and reaction motif SpyTag, wherein the connecting part of the BDTag and the DHFR is inserted with the enzyme cutting site of TVMV enzyme. The corresponding amino acid sequence of BDTag-DHFR-SpyTag is SEQ ID NO: 7, wherein the 6-11 amino acid sequence is a 6 XHis tag, the 14-42 amino acid sequence is BDTag, the 47-53 amino acid residues are TVMV enzyme recognition sites, the 66-224 amino acid sequence is target protein DHFR, and the 237-249 amino acid sequence is SpyTag.
(d) c-SpyStapler-GFPrm: the original translated amino acid sequence is SEQ ID NO: 8, from the N end to the C end, an intein C-terminal part, a response motif SpyStapler, a target protein GFPrm and a separated intein N-terminal part are respectively arranged, wherein 5-43 th amino acid sequence is the separated intein C-terminal part, 46-51 th amino acid sequence is a 6 XHis tag, 59-119 th amino acid sequence is the response motif SpyStapler, 132-369 th amino acid sequence is the target protein GFPrm, 382-388 th amino acid residue is a TEV enzyme digestion site, and 389-490-th amino acid sequence is the separated intein N-terminal part. After the gene sequence is translated, two parts of separated inteins react and are subjected to self-shearing, so that the cyclic c-SpyStapler-GFPrm is obtained.
The method utilizes conventional characterization means, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography mass spectrometry (LC-MS), matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) and orthogonal enzymolysis reaction to carry out basic characterization on the obtained sorbite structure and indirect demonstration on the topological structure of the sorbite structure.
The invention utilizes fluorescence spectrum to explore the influence of the soxhlet hydrocarbon on the performance of target protein, and utilizes Differential Scanning Calorimetry (DSC) to carry out melting temperature (T) on the obtained soxhlet hydrocarbon structurem) And testing and exploring the influence of the solvolysis on the thermal stability of the target protein. The present invention then explores the enzymatic resistance of hydrocarbon structures using a trypsin enzymatic reaction. Then, the enzymatic activity of the catenane cat-GFPrm-DHFR obtained by the reaction of BD-DHFR-A and c-S-GFPrm was tested, the enzymatic activity retention capability of the catenane structure after heating at 50 ℃ for A certain time was investigated, and the influence of the mechanical denaturation introduced by freeze-thawing on the enzymatic activity was investigated by means of freeze-thawing.
The invention inserts the sequences of c-SpyStapler-ELP1 (noted as c-SpyStapler-ELP1, abbreviated as c-S-E1) and BD-E2-A without 6 × His tag into pACYCDuet-1 co-expression vector to obtain a heterosuxohydrocarbon structure co-expression system, and the heterosuxohydrocarbon structure co-expression system can realize the direct cell synthesis of protein heterosuxohydrocarbon by transforming the heterosuxohydrocarbon structure co-expression system into escherichia coli BL21(DE3) competent cells.
The invention optimizes the active template AT-Spy based on the SpyTag-SpyCatcher compound, and refers to a SpyTag-SpyCatcher 002 system (Keeble, A.H., et al, Angew.Chem.int.Ed.2017,56, 16521-16525) with better reaction efficiency reported in the literature to obtain the active template AT-Spy002 (namely comprising BDTag002, SpyStapler002 and SpyTag 002) with better reactivity, and the active template AT-Spy002 system is applied to the preparation of target elastin-like hydrocarbon, so that the AT-Spy002 system has better reactivity compared with AT-Spy, and the prepared heterogeneous hydrocarbon structure has better enzymolysis resistance.
The structure of the reaction precursor in the preparation of the protein catenane structure from the AT-Spy002 system is illustrated below by specific examples:
(a) BDTag-ELP2-SpyTag 002: from the N end to the C end, the DNA fragment is respectively a 6 XHis tag, a reaction motif BDTag002, a target protein ELP2 and a reaction motif SpyTag002, wherein the connecting part of the BDTag002 and the ELP2 is inserted with an enzyme cutting site of TVMV enzyme. The corresponding amino acid sequence of BDTag-ELP2-SpyTag 002 is SEQ ID NO: 9, wherein the 6-11 amino acid sequence is a 6 XHis tag, the 14-61 amino acid sequence is BDTag002, the 70-76 amino acid residue is a TVMV enzyme recognition site, the 79-160 amino acid sequence is a target protein ELP2, and the 163-176 amino acid sequence is SpyTag 002.
(b) c-SpyStapler002-ELP 1: the original translated amino acid sequence is SEQ ID NO: 10, from the N end to the C end, an intein C-terminal part, a target protein ELP1 ', a reaction motif SpyStapler002, a target protein ELP1 ' and a separated intein N-terminal part are respectively arranged, wherein the amino acid sequence at the 5-43 position is the separated intein C-terminal part, the amino acid sequence at the 46-51 position is a 6 XHis tag, the amino acid sequence at the 54-78 position is a first section of ELP (ELP1 '), the amino acid sequence at the 81-143 position is the reaction motif SpyStapler002, the amino acid sequence at the 146- & gtand 245 position is a second section of the target protein ELP (ELP1), the amino acid residue at the 248- & gtand 254 position is a TEV enzyme digestion site, and the amino acid sequence at the 255- & gtand 356 position is a separated intein N-terminal part. After the gene sequence is translated, two parts of separated inteins react and are subjected to self-shearing, so that the cyclic c-SpyStapler002-ELP1 is obtained.
According to the invention, each secondary component (SpyTag, BDTag and SpyStapler) in the SpyTag-SpyCatcher compound is re-connected, a chain entanglement structure is introduced, a protein active template method combining chain entanglement and catalysis is developed, and mechanical bond coupling of different target proteins connected in a catenane form can be realized. The method has the advantages of realizing the concise synthesis of protein heterosoxohydrocarbon and providing a novel protein coupling strategy. The method does not affect the structure and activity preparation of the target protein, and simultaneously improves the enzymolysis resistance, heat-resistant induced denaturation resistance and mechanical denaturation resistance of the target protein. Meanwhile, the soxhlet alkylation process can realize direct expression preparation in cells through a co-expression system. In addition, the active template can be optimized, and the reaction efficiency and the property of the protein conjugate are further improved.
Drawings
FIG. 1 shows the design of the active template of the present invention and the use of the active template to achieve heterogeneous solenoalkylation of a protein of interest.
FIG. 2 shows mass spectra data for each of the reaction precursors BD-A (a), c-S (b), BD-E2-A (c), c-S-E1(d), BD-DHFR-A (E), and c-S-GFPrm (f) of example 2.
FIG. 3 shows a SDS-PAGE data of the synthesis of the proteins cat-spy (a), cat-ELP (b) and cat-GFPrm-DHFR (c) in example 3.
FIG. 4 shows mass spectral data and size exclusion chromatographic data for the proteins catenane cat-Spy (a, b), cat-ELP (c, d) and cat-GFPrm-DHFR (e, f) in example 3.
FIG. 5 shows the results of the cleavage experiments of the proteins cat-spy (b), cat-ELP (c) and cat-GFPrm-DHFR (d) TEV protease and TVMV protease in example 4, and the schematic diagram of the cleavage structure prediction (a).
FIG. 6 shows a chart of DSC data for cat-spy (a), cat-ELP (b), GFPrm (c), c-S-GFPrm (d) and cat-GFPrm-DHFR (e) in example 5.
FIG. 7 shows fluorescence spectra of GFPrm, c-S-GFPrm, cat-GFPrm-DHFR in example 5.
FIG. 8 shows the tryptic datA of BD-DHFR-A, c-S-GFPrm, cat-GFPrm-DHFR in example 5.
FIG. 9 shows the enzyme activity of wild-type DHFR, BD-DHFR-A, cat-GFPrm-DHFR in example 5 after heating at 50 ℃ for A certain period of time (A) and the enzyme activity of DHFR before and after freeze-thawing (b).
FIG. 10 shows the standard curve of NADPH absorbance in example 5 (A), and the kinetic datA of DHFR, BD-DHFR-A, cat-GFPrm-DHFR after heating at 50 ℃ for A certain period of time (b-d), and (e) reduction experiments on dihydrofolate before and after freeze-thawing.
FIG. 11 shows a schematic diagram of the procedure for preparing a catenane structure by direct intracellular expression in example 6 (a), and a data diagram of SDS-PAGE (b), a data diagram of size exclusion chromatography (c), and a data diagram of matrix assisted laser desorption-time of flight mass spectrometry (d) of the catenane structure obtained by direct intracellular expression and its protease digestion experiment.
FIG. 12 shows the SDS-PAGE data of the reaction of the active template resolved from the structure of the Spy002 complex obtained by optimizing the structure of the original Spy complex in example 7 in the preparation of the catenated protein structure (a), the results of the digestion experiment of the catenated protein structure (b), the reaction efficiency comparison of the active template resolved from the original Spy complex (AT-Spy) and the active template resolved from the optimized structure Spy002 accord (AT-Spy002) AT different reaction concentrations (c) or different reaction temperatures (d), and the trypsin enzymolysis of the catenated hydrocarbon structures cat-ELP (e) and cat-ELP002(f) respectively prepared from AT-Spy and AT-Spy 002.
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 specific steps for preparing the precursor of the protein heterogeneous catenane structure are as follows: construction of His-containing recombinant gene engineering technology6A tag (for protein purification), BDTag (reaction motif), target protein elastin (ELP2) (or other proteins such as DHFR, or without target protein), and a fusion protein of SpyTag (reaction motif), namely a gene sequence of BDTag-ELP2-SpyTag (or BDTag-DHFR-SpyTag, BDTag-SpyTag), and inserting the gene sequence into an expression vector pQE-80L; and construction of a peptide containing the C-terminal portion of the isolated intein (Int)C) SpyStapler (reaction motif), elastin protein of interest (ELP1) (either other protein such as GFPrm or not added), and isolated intein N-terminal moiety (Int)N) That is, the gene sequence of c-SpyStapler-ELP1 (or c-SpyStapler-GFPrm, c-SpyStapler) was inserted into expression vector pET-15 b. Then the expression vector is transformed into escherichia coli for expression, and a purified target reaction precursor protein is obtained by utilizing protein purification methods such as Ni affinity chromatography and the like. The fusion protein of interest is, for example: BDTag-ELP2-SpyTag (BD-E2-A), BDTag-DHFR-SpyTag (BD-DHFR-A), BDTag-SpyTag (BD-A), c-SpyStapler-ELP1(c-S-E1), c-SpyStapler-GFPrm (c-S-GFPrm), c-SpyStapler (c-S) and the like, wherein ELP1 and ELP2 refer to elastin-like proteins with different lengths.
Performing primary characterization on precursor proteins and prepared soxhlet hydrocarbon structures by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), liquid mass spectrometry (LC-MS), matrix analysis laser-assisted time-of-flight mass spectrometry (MALDI-TOF-MS) and Size Exclusion Chromatography (SEC); enzyme digestion experiments of TEV enzyme and TVMV enzyme are combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to indirectly prove the topological structure of the protein catenane; the influence of the charged hydrocarbons on the structure of the folded protein is characterized by using a fluorescence spectrum test; subjecting the obtained catenane structure to T by Differential Scanning Calorimetry (DSC)mAnd testing and exploring the influence of the solvolysis on the thermal stability of the target protein. The present invention then explores the enzymatic resistance of hydrocarbon structures using a trypsin enzymatic reaction. Then, the enzymatic activity of the catenane cat-GFPrm-DHFR obtained by the reaction of BD-DHFR-A and c-S-GFPrm was tested, the enzymatic activity retention capability of the catenane structure after heating at 50 ℃ for A certain time and the mechanical change introduced by freeze thawing were investigated by freeze thawing meansInfluence of sex on the enzyme activity.
Example 1: expression and purification of reaction precursor protein
During construction, reaction precursors BD-A, BD-ELP2-A and BD-DHFR-A are constructed in pQE-80L vector, and c-S, c-S-E1 and c-S-GFPrm are constructed in pET-15b vector. The vector was transformed into E.coli BL21(DE3) competent cells and cultured overnight. Subsequently, the colonies grown on the plate were selected and inoculated into 5mL of LB medium containing 100. mu.g/mL of ampicillin sodium, and cultured at 37 ℃ for 8 to 12 hours. Then transferring the obtained seed liquid into 1L fresh LB culture medium at a ratio of 1:100 or 1:200, and performing shake culture at 37 deg.C to OD600Between 0.6 and 0.8, isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM, and the expression was carried out for 20 hours at 16 ℃. The cells were collected by centrifugation. The cells were then dispersed in 30-35mL of lysis buffer A (50mM disodium phosphate, 300mM sodium chloride, 10mM imidazole, pH 8.0), disrupted by ultrasonication, centrifuged (12000 g.times.30 min), and the supernatant was incubated with a nickel affinity resin at 4 ℃ for 1 hour. Subsequently, the mixed liquid was poured into an empty gravity column, washed 5 to 10 column volumes with buffer B (50mM disodium hydrogenphosphate, 300mM sodium chloride, 20mM imidazole, pH 8.0), and then eluted with 2 column volumes of eluent C (50mM disodium hydrogenphosphate, 300mM sodium chloride, 250mM imidazole, pH 8.0). The eluate was further purified by separation using AKTA protein purification system (AKTA Avant, GEHealthcare) with PBS buffer (pH7.4) as a mobile phase at a flow rate of 0.5mL/min, and a sample was collected for the preparation of the catenane structure.
Example 2: characterization of reaction precursors
And carrying out primary characterization on the purified reaction precursor by using high performance liquid chromatography-electrospray mass spectrometry. FIG. 2 shows mass spectra data of BD-A (a), c-S (b), BD-E2-A (c), c-S-E1(d), BD-DHFR-A (E), and c-S-GFPrm (f) precursors for preparing protein heterosoxhlet structures, wherein experimental data substantially matches theoretical calculation data.
Example 3: preparation and characterization of protein heterohydrocarbons
The purified reaction precursors BD-A/BD-E2-A/BD-DHFR-A and c-S/c-S-E1/c-S-GFPrm were mixed at A reaction concentration of 50. mu.M at A ratio of 1:1, while 10mM Dithiothreitol (DTT) was added to the system (5 mM DTT was added to the reaction system BD-DHFR-A and c-S-GFPrm) to reduce and separate intermolecular disulfide bonds. The mixed system is reacted for 20h at 4 ℃. After the reaction, 10. mu.L of the reaction precursor and the reaction mixture were mixed with 5 Xprotein loading buffer, followed by heating at 98 ℃ for 10min, and analysis of the results was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fig. 3 shows the reaction of these three pairs of reactive precursors, in which a new band with a molecular weight corresponding to the sum of the two reactive precursors appears in the reaction mixture, that is to say a catenane structure is formed. The reacted mixture is then separated by size exclusion chromatography, and the fractions of the hydrocarbons are collected and analyzed for molecular weight using high performance liquid chromatography-electrospray mass spectrometry (LC-MS) or matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS). In FIG. 4, (A), (c), (E) are the relevant mass spectrA datA of the catenane structure cat-Spy formed by the reaction of BD-A and c-S, respectively, the catenane structure cat-ELP formed by the reaction of BD-E2-A and c-S-E1, and the catenane structure cat-GFPrm-DHFR formed by the reaction of BD-DHFR-A and c-S-GFPrm, respectively, the test results are consistent with the theoretical calculation datA, while in FIG. 4, (b), (d), (f) are the size exclusion chromatography datA of the corresponding reaction precursor and the catenane structure, respectively, which indicate that the resulting reaction structure has A larger hydrodynamic system compared to the reaction precursor (for cat-Spy, the outflow volume is close to that of the reaction precursor BD-A due to the tighter structure after the reaction), further proves that the reaction precursor reacts to obtain the catenane structure.
Example 4: topology characterization of protein heterohydrocarbons
After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography-electrospray mass spectrometry (LC-MS) or matrix assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) and size exclusion chromatography are used for verifying that reaction can occur between reaction precursors, the invention indirectly proves that a structure formed after reaction is a sorbite structure by utilizing an orthogonal enzyme digestion experiment and combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
In the process of precursor molecule construction, A Tobacco Vein Mottle Virus (TVMV) protease enzyme cutting site is added into BD-A/BD-E2-A/BD-DHFR-A, and A Tobacco Etch Virus (TEV) protease is inserted into c-S/c-S-E1/c-S-GFPrm. In the process of enzyme digestion experiment, reaction precursors BD-A/BD-E2-A/BD-DHFR-A and TVMV protease are mixed according to the concentration ratio of enzyme to substrate of 1:5, and enzyme digestion is carried out for 4h at 37 ℃. Mixing the reaction precursor c-S/c-S-E1/c-S-GFPrm and TEV protease according to the concentration ratio of enzyme to substrate of 1:20, and carrying out enzyme digestion at 37 ℃ for 4 h. The catenane structure cat-Spy/cat-ELP/cat-GFPrm-DHFR was subjected to 3 sets of enzymatic cleavage experiments: (1) the soxhlet hydrocarbon structure is only mixed with TEV protease according to the concentration ratio of enzyme to substrate of 1: 20; (2) the sorbite structure is only mixed with TVMV protease according to the concentration ratio of enzyme to substrate of 1: 5; (3) mixing the sorbite structure with TEV protease and TVMV protease respectively according to the proportion; all 3 groups of enzyme cutting systems are cut for 4 hours at 37 ℃. And mixing the enzyme digestion product and the substrate before enzyme digestion with 5 multiplied by protein loading buffer solution respectively, heating at 98 ℃ for 10min, and performing result analysis by utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FIG. 5 shows the theoretical cleavage product of the catenane structure and the actual cleavage results. The reaction precursor BD-A/BD-E2-A/BD-DHFR-A has A linear structure before enzyme digestion, has two linear structures after enzyme digestion by TVMV, and shows two lower bands or A lower main band in an SDS-PAGE (for the BD-A system, the difference is not shown in an SDS-PAGE datA diagram due to smaller molecular weight); the reaction precursor c-S/c-S-E1/c-S-GFPrm has a cyclic structure before enzyme digestion and a linear structure after enzyme digestion, and the data graph of SDS-PAGE shows that the linear structure has larger apparent molecular weight compared with the cyclic structure. The catenane structure, when cleaved with TEV protease alone, gives linear l-S/l-S-E1/l-S-GFPrm (l indicates linear structure) and cyclic c-BD-A/c-BD-E2-A/c-BD-DHFR-A (c indicates linear structure); when only TVMV protease is used for enzyme digestion, cyclic c-S/c-S-E1/c-S-GFPrm and linear l-BD-A/l-BD-E2-A/l-BD-DHFR-A can be obtained; the l-S/l-S-E1/l-S-GFPrm and l-BD-A/l-BD-E2-A/l-BD-DHFR-A, which were linear when cleaved simultaneously with TEV protease and TVMV protease, exhibited different apparent molecular weights in SDS-PAGE. As can be seen in fig. 5, the experimental results are consistent with the predicted results.
Example 5: characterization of protein heterosoxhlet hydrocarbon structural properties
After preparing and obtaining the catenane structures cat-Spy, cat-ELP and cat-GFPrm-DHFR and proving the topological structures, the invention carries out property exploration on the catenane structures.
Differential Scanning Calorimetry (DSC) was first used to explore the thermal stability of the hydrocarbon structure conjugates. The samples were diluted to a concentration of 1mg/mL and tested using a MicroCal VP-DSC (GE Healthcare) in which the samples were scanned from 30 ℃ to 110 ℃ at a rate of 2 ℃ per minute, and the data obtained were processed through a MicroCal Analysis launcher (GE Healthcare). FIG. 6 shows the DSC curve obtained, and Table 1 shows T of the structure obtainedmData tables, and data reported in the literature for the SpyTag-BDTag/SpyStapler complex and DHFR. The data in Table 1 show that the prepared catenane structure has no significant effect on the thermal stability of the target protein. In terms of thermostability, the stability of the entangled structure formed by the active template is lower than that of the target protein, and the entangled structure does not function as a domain limiter, so that the thermostability of the protein cannot be improved.
TABLE 1
aFinger TmThe data were obtained from a temperature-variable CD test,bfinger TmThe data were obtained from a DSC test,cfinger TmData are reported in the literature.
[1]Wu,X.L.；Liu,Y.；Liu,D.；Sun,F.；Zhang,W.B.,J.Am.Chem.Soc.2018,140,17474-17483.
[2]Tian,J.；Woodard,J.C.；Whitney,A.；Shakhnovich,E.I.,PLOSComput.Biol.2015,11,e1004207-e1004233.
Subsequently, the present study explored the effect of catenane structure on protein structure using fluorescence spectroscopy. GFPrm, c-S-GFPrm and cat-GFPrm-DHFR are used for fluorescence spectroscopy. The samples were diluted with a buffer (PBS, 5mM DTT, pH7.4) and added to a black 96-well plate for data acquisition using a microplate reader (Perkinelmer Co.) scanning mode. In the test process, fixing 540nm as the emission wavelength, and scanning the excitation spectrum of 400-520 nm; then, the excitation wavelength was fixed at 440nm, and the emission spectrum at 490-600nm was scanned. FIG. 7 is a fluorescence spectrum superposition spectrum of three samples, GFPrm, c-S-GFPrm and cat-GFPrm-DHFR, which are basically identical, showing that the coupling achieved by means of soxhlet alkylation does not affect the structure of the target protein.
In addition, the present invention has performed trypsin enzymolysis experiments on the catenane structure. The reaction precursor BD-DHFR-A, c-S-GFPrm and the catene structure cat-DHFR-GFPrm (20 μ M, PBS buffer) were mixed with trypsin working solution (100 μ g/mL, 4 μ M) at a concentration of 100:1, and the mixture was subjected to enzymatic hydrolysis at 22 ℃. Then, at specific time intervals (10, 20, 30, 40, 50, 60, 120, 180, 240min), 20. mu.L of the mixture was mixed with 5 Xprotein loading buffer for reaction quenching, and analyzed by SDS-PAGE, followed by relative quantification of the band of interest by image J. FIG. 8 shows the results of enzyme digestion experiments, and the catenane structure has better enzymolysis resistance compared with linear and cyclic reaction precursors.
Finally, the present invention has been made to investigate the activity of dihydrofolate reductase (DHFR) which is a target protein in a catenane structure. DHFR, BD-DHFR-A and cat-DHFR-GFPrm were diluted in KHP buffer (40.1mM dipotassium hydrogenphosphate, 9.9mM sodium dihydrogenphosphate, 5mM mercaptoethanol, pH7.4) to A final concentration of 100nM to obtain A working solution of the substrate. The working solution was mixed with 100. mu.M ADPH (KHP buffer) and 100. mu.M dihydrofolate (KHP buffer) and placed in a 96-well plate, followed by real-time tracking of absorbance changes at 340nm using a microplate reader (PerkinElmer corporation) kinetic mode. In addition, the absorbance at 340nm of KHP buffers containing different concentrations of NADPH (10, 20, 30, 40, 50, 60, 100nmol) was tested for plotting a standard curve. The resulting absorbance-time curve of the sample was taken for analysis in a linear portion and the enzyme activity was calculated in combination with the standard curve. From fig. 9 and 10, it can be seen that the DHFR enzyme activity of the catenane structure is substantially the same as that of the wild-type DHFR, but the activity of the reaction precursor BD-DHFR- A is greatly lost, which is probably because the addition of peptide fragments at both ends in the reaction precursor affects the DHFR structure, and after the catenane structure is formed, the added peptide fragments participate in the formation of the middle Spy domain, so that the influence on the DHFR structure is reduced, and the DHFR enzyme activity is recovered. Then, the samples DHFR, BD-DHFR-A and cat-DHFR-GFPrm working solution were heated at 50 ℃ for A certain period of time (10, 20, 30, 40min), and immediately placed at 4 ℃, and then enzyme activity analysis was performed according to the above steps, and the results are shown in FIG. 9 (A) and FIG. 10. DHFR and BD-DHFR-A have certain enzyme activity reduction after heating, and the catenane structure maintains the activity to A greater extent. In addition, the present invention also explores the effect of freeze-thawing on the activity of the target protein. Samples DHFR, BD-DHFR-A and cat-DHFR-GFPrm working solution were frozen at-80 ℃ and then rapidly thawed at room temperature, and then subjected to enzyme activity analysis according to the above activity test procedure, and the results are shown in FIG. 9 (b) and FIG. 10, in which the catenane structure largely retains the enzyme activity, while the wild-type DHFR activity is largely reduced.
The above results show that the coupling of different proteins by means of a catenane structure does not affect the structure and activity of the proteins, but reduces the influence of the added peptide fragments on the enzyme activity due to the formation of the intermediate entanglement domain (i.e. the Spy domain obtained by the structural recombination of the reaction motifs BDTag, SpyTag and SpyStapler). Meanwhile, compared with a linear structure and a ring structure, the catenane structure has better enzymolysis resistance, heat-resistant induced denaturation resistance and mechanical denaturation resistance introduced by freeze thawing under certain conditions.
Example 6: preparation of protein heterosoxohydrocarbons by direct intracellular expression
After extracellular synthesis of the protein catenane structure was achieved, the present invention recombined genes encoding reaction precursors BD-E2-a and c-S-E1 (lacking only 6 × His tag compared to c-S-E1), respectively, into the co-expression vector pacycdue-1. The expression vector was subsequently transformed into E.coli BL21(DE3) competent cells and cultured overnight. Subsequently, the colonies grown on the plate were selected and inoculated into 5mL of LB medium containing 25. mu.g/mL of chloramphenicol, and cultured at 37 ℃ for 8 to 12 hours. Then, the obtained seed liquid is mixed according to the proportion of 1:100 portions of the culture were transferred to 1L of fresh LB medium and cultured with shaking at 37 ℃ to OD600Adding 0.6-0.8 of isopropyl-B-D-thiogalactopyranoside (IPTG) was brought to a final concentration of 1mM and expressed at 16 ℃ for 20 hours. The cells were collected by centrifugation. Then, the mixture is purified according to the example 1, and a certain amount of the mixture is taken out for TEV enzyme and/or TVMV enzyme digestion experiments after the purification through size exclusion chromatography, and the experimental steps are the same as the example 4. And then mixing the obtained sorbite structure and the enzyme digestion product with a 5 multiplied protein loading buffer solution for SDS-PAGE analysis, wherein the sorbite can be prepared by direct cell expression of a co-expression system as shown in figure 11(b), and meanwhile, the enzyme digestion experiment proves the topological structure. Size exclusion chromatography (FIG. 11c) also showed that the main product was a soxhlet structure after co-expression of the system. And then carrying out matrix-assisted laser desorption-time-of-flight mass spectrometry on the obtained sorbite structure, wherein the test data is consistent with theoretical calculation data as shown in figure 11(d), and the fact that the in vivo direct expression preparation of the protein sorbite structure can be realized is proved.
Example 7: optimization of active templates
After the bioactive template is prepared by re-connecting the SpyTag-SpyCatcher compound, the bioactive template is optimized by the method so as to prepare the bioactive template with better reactivity. In the previous literature report, the SpyTag-SpyCatcher 002 complex is a protein reaction pair with better reactivity obtained by performing directed evolution on the basis of the original SpyTag-SpyCatcher complex (Keeble, a.h., et al, angelw.chem.int.ed.2017, 56, 16521-16525), and in view of the above, the SpyTag-SpyCatcher 002 complex and the original SpyTag-SpyCatcher complex are generally re-connected to prepare the active template AT-Spy002 with better reaction efficiency, and the reaction precursors are BDTag-ELP2-SpyTag 002, c-ELP-spystatcler 002. The reaction precursor was then expressed and purified using the method described in example 1, and a heterosoxhlet hydrocarbon structure was prepared using the method described in example 3 (fig. 12 (a)). After preparation of the resulting soxhlet structure, the formation of a heterogeneous soxhlet structure was further confirmed using TEV enzyme and/or TVMV enzyme digestion experiments (fig. 12 (b)). Meanwhile, in the research, the reaction conditions of the active template AT-Spy002 under different conditions were investigated by changing the reaction concentration and the reaction temperature of the reaction precursor and the reaction efficiency was compared with the original reaction template AT-Spy, and it was found that the active template AT-Spy002 has better reactivity than AT-Spy (FIG. 12(c, d)). In addition, the invention carries out trypsin digestion experiment on the soxhlet hydrocarbon structures (cat-ELP and cat-ELP002) of the ELP prepared by two active templates, and the experiment proves that the cat-ELP002 prepared by the AT-Spy002 has better enzymolysis resistance compared with the cyclic precursor and the cat-ELP (figure 12(e, f)).
SEQUENCE LISTING
<110> Beijing university
<120> protein coupling method based on soyabean alkylation
<130>WX2019-03-091
<160>13
<170>PatentIn version 3.5
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Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala
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Lys Thr Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
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Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
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Asp Gly Pro Val Arg Leu Pro Asp Asn His Tyr Leu Leu Thr Gln Ser
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Val Ile Ser Lys Asp Pro Asn Glu Lys Arg Asp His Ala Val Leu His
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Glu Asp Gly Ser Leu Ile Arg Ala Thr Lys Asp His Lys Phe Met Thr
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Val Asp Gly Gln Met Leu Pro Ile Asp Glu Ile Phe Glu Arg Glu Leu
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Asp Leu Met Arg Val Asp Asn Leu Pro Asn
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Gly Thr Val Pro Thr Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys
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His His His Glu Leu Gly His Gly Val Gly Val Pro Gly Val Gly Val
50 55 60
Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly His Met
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Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly His Val Lys Asp
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Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro
100 105 110
Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln
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Gly Gln Val Thr Val Asn Gly Glu Ala Thr Lys Gly Asp Ala His Thr
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Asp Gly Pro Gln Gly Ile Trp Gly Gln Thr Ser Val Pro Gly Val Gly
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Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val
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Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
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Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
195 200 205
Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val
210 215 220
Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu Leu Asp Gly Pro Gln
225 230 235 240
Gly Ile Trp Gly Gln Gly Thr Glu Asn Leu Tyr Phe Gln Gly Cys Leu
245 250 255
Ser Tyr Glu Thr Glu Ile Leu Thr Val Glu Tyr Gly Leu Leu Pro Ile
260 265 270
Gly Lys Ile Val Glu Lys Arg Ile Glu Cys Thr Val Tyr Ser Val Asp
275 280 285
Asn Asn Gly Asn Ile Tyr Thr Gln Pro Val Ala Gln Trp His Asp Arg
290 295 300
Gly Glu Gln Glu Val Phe Glu Tyr Cys Leu Glu Asp Gly Ser Leu Ile
305 310 315 320
Arg Ala Thr Lys Asp His Lys Phe Met Thr Val Asp Gly Gln Met Leu
325 330 335
Pro Ile Asp Glu Ile Phe Glu Arg Glu Leu Asp Leu Met Arg Val Asp
340 345 350
Asn Leu Pro Asn
355
<210>11
<211>48
<212>PRT
<213> Artificial sequence
<400>11
Val Thr Thr Leu Ser Gly Leu Ser Gly Glu Gln Gly Pro Ser Gly Asp
1 5 10 15
Met Thr Thr Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg
20 25 30
Asp Glu Asp Gly Arg Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp
35 40 45
<210>12
<211>14
<212>PRT
<213> Artificial sequence
<400>12
Val Pro Thr Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys
1 5 10
<210>13
<211>73
<212>PRT
<213> Artificial sequence
<400>13
Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly His Val Lys Asp
1 5 10 15
Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro
20 25 30
Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln
35 40 45
Gly Gln Val Thr Val Asn Gly Glu Ala Thr Lys Gly Asp Ala His Thr
50 55 60
Asp Gly Pro Gln Gly Ile Trp Gly Gln
65 70
Claims (7)
1. A method for soxhlation-based protein coupling comprising the steps of:
1) designing two precursor sequences of protein catenane reaction, namely a circular fusion protein sequence of SpyStapler-target protein 1 and a linear fusion protein sequence of BDtag-target protein 2-Spytag;
2) constructing coding genes corresponding to the two precursor sequences in the step 1), and introducing a vector plasmid;
3) expressing two reaction precursors in different cells respectively, purifying the two reaction precursors respectively, mixing the two reaction precursors in vitro under a certain condition, and carrying out structural recombination on the two reaction precursors in the process so as to form isopeptide bonds in situ and realize the preparation of a heterogeneous catenane structure; or co-expressing two reaction precursors in the cell, carrying out structural recombination on the two reaction precursors in the cell, further forming isopeptide bonds in situ, realizing the preparation of the heterogeneous sorbite structure, and then purifying the protein with the heterogeneous sorbite structure.
2. The protein coupling method according to claim 1, wherein the amino acid sequences of BDTag, SpyTag and SpyStapler in step 1) are respectively as shown in SEQ ID NOs: 3. SEQ ID NO: 1 and SEQ ID NO: 4, or a mutant thereof which retains coupling reactivity; the amino acid sequences of BDTag, SpyTag and SpyStapler mutants are respectively shown as SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: shown at 13.
3. The protein coupling method according to claim 1, wherein in step 1), the C-terminal portion and the N-terminal portion of the isolated intein are fused to the N-terminus and the C-terminus of the spystpler-target protein 1 fusion protein, respectively.
4. The protein coupling method as set forth in claim 1, wherein step 1) is to design a His-tag sequence at the N-terminus of the fusion protein, and in step 3) the protein is purified by nickel affinity chromatography.
5. The protein coupling method according to claim 1, wherein the encoding genes corresponding to the circular SpyStapler-target protein 1 fusion protein sequence and the linear BDTag-target protein 2-SpyTag fusion protein sequence are constructed on expression vectors in step 2) separately or on the same co-expression vector.
6. The protein conjugation method of claim 1, wherein the target protein 1 and the target protein 2 are two different proteins.
7. The protein coupling method according to claim 6, wherein the fusion protein sequence of the circular SpyStapler-target protein 1 is shown as SEQ ID NO: 6, the linear BDtag-target protein 2-Spytag fusion protein has a sequence shown as SEQ ID NO: 5 is shown in the specification; or, the fusion protein sequence of the circular SpyStapler-target protein 1 is shown as SEQ ID NO: 8, the linear BDtag-target protein 2-Spytag fusion protein has a sequence shown as SEQ ID NO: shown at 7.
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| CN113045633B (en) * | 2019-12-27 | 2022-04-26 | 北京大学 | Design of protein heterogeneous entanglement primitive and preparation method of complex catenane structure |
| CN111560391B (en) * | 2020-05-21 | 2022-02-11 | 北京大学 | Biosynthesis method of protein heterogeneous catenane |
| KR102547393B1 (en) * | 2021-04-19 | 2023-06-23 | 엠브릭스 주식회사 | Protein Complex Comprising Botulinum Toxin Translocation Domain and Endolysin, and Anti-Bacterial Composition Comprising Thereof |
| CN114075298B (en) * | 2022-01-07 | 2022-04-29 | 广州中科蓝华生物科技有限公司 | Soxhydrogenated VAR2CSA recombinant protein and preparation method and application thereof |
| CN116621947B (en) * | 2023-07-18 | 2023-11-07 | 北京智源人工智能研究院 | Topological protein based on Soxhlet skeleton, preparation method and application |
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