WO2023071267A1 - 基于大肠杆菌素e家族dna酶的蛋白质复合物及其在人工蛋白支架中的应用 - Google Patents

基于大肠杆菌素e家族dna酶的蛋白质复合物及其在人工蛋白支架中的应用 Download PDF

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WO2023071267A1
WO2023071267A1 PCT/CN2022/102567 CN2022102567W WO2023071267A1 WO 2023071267 A1 WO2023071267 A1 WO 2023071267A1 CN 2022102567 W CN2022102567 W CN 2022102567W WO 2023071267 A1 WO2023071267 A1 WO 2023071267A1
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protein
seq
scaffold
amino acid
acid sequence
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马立新
翟超
杨军
王飞
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湖北大学
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Definitions

  • the invention belongs to the field of synthetic biology and nanobiology technology, and in particular relates to a protein complex based on colicin E family DNA enzymes and its application in artificial protein scaffolds.
  • High-affinity protein interaction pairs are widely used in many fields, such as co-immunoprecipitation, protein pull-down, yeast two-hybrid and enzyme immobilization, etc., in which the multi-enzyme complex formed by self-assembly of protein interaction
  • the system has attracted much attention due to its excellent synergistic catalytic ability.
  • multiple orthogonal protein interaction pairs are used to assemble multiple enzyme molecules in a specific order, and cascade catalytic reactions are performed in the form of artificial multi-enzyme complexes. It is conducive to the formation of substrate channels, which can prevent the diffusion of intermediate products and promote the timely transformation of intermediate products, thereby avoiding the interference of certain toxic intermediates on enzyme activity and improving reaction efficiency.
  • the protein-protein interaction is mainly realized through the basic assembly element Cohesin-Dockerin derived from the cellulosome and artificially designed protein interaction pairs such as SpyCatcher/SpyTag, SnoopCatcher/SnoopTag and RIAD/RIDD.
  • the existing protein interaction pairs have many problems in practical application. First of all, the number of strong interaction pairs is limited, and the affinity of commonly used interaction pairs is not strong. When constructing more complex multi-enzyme complexes, the structure is unstable and easy to collapse. Secondly, for some exothermic reactions, such as the degradation of cellulose, thermostable protein interaction pairs are required to combine with thermostable enzymes to construct multi-enzyme complexes, and such protein interaction pairs are even rarer at present.
  • Colicin produced by E. coli, is a cytotoxin that kills closely related bacilli.
  • the DNases of the colicin E family include CE2, CE7, CE8, and CE9, which are non-specific endonucleases with highly similar structures.
  • CE proteins combine with their corresponding immune proteins Im2, Im7, Im8, and Im9 to inhibit the binding of CE proteins to DNA, thereby eliminating their toxicity to the host.
  • the combination of CE protein and Im protein is one of the strongest protein interactions known in nature, with a K d of 10 -14 to 10 -17 .
  • the combination of Im and CE is a "double recognition mechanism", that is, the Im protein first forms a sequence-independent complex with the CE nuclease through the conserved element (helix III); then the helix II containing specific residues on the Im protein binds to the corresponding CE Nuclease binding, stabilizing the orthogonal complex (Li W., et al, Biochemistry. 1998, 37, 11771-11779; Keeble AH, Kleanthous C., J Mol Biol. 2005, 352, 656-671).
  • the object of the present invention is to provide the protein complex based on the colibactin E family DNase and its application in the artificial protein scaffold, the present invention is through the carboxyl terminal DNase structure domain of the DNase CE2, CE7, CE8 and CE9 of CE family Protein engineering was carried out to obtain mutants that lost DNase activity but still retained super high affinity to the corresponding Im protein, and constructed CL2/Im2, CL7/Im7, CL8/Im8 and CL9/Im9 protein interaction pairs. Studies have found that these protein interaction pairs have properties such as heat resistance, high affinity, high specificity, small molecular weight, and fast assembly speed. Based on this, artificial protein scaffolds were constructed, and a new platform for the construction of artificial multi-enzyme complexes was built.
  • One of the objectives of the present invention is to provide a protein complex based on colibactin E family DNA enzymes, the protein complex includes: the interaction pair formed by CL2 protein and Im2 protein, or the interaction pair formed by CL7 protein and Im7 protein An interaction pair, or any one or more of the interaction pair formed by CL8 protein and Im8 protein, or the interaction pair formed by CL9 protein and Im9 protein; wherein,
  • the amino acid sequence of the CL2 protein is shown in SEQ ID NO.2;
  • the amino acid sequence of the CL7 protein is shown in SEQ ID NO.5;
  • the amino acid sequence of the CL8 protein is shown in SEQ ID NO.8;
  • the amino acid sequence of the CL9 protein is shown in SEQ ID NO.11.
  • the amino acid sequence of the Im2 protein is shown in SEQ ID NO.3;
  • the amino acid sequence of the Im7 protein is shown in SEQ ID NO.6;
  • the amino acid sequence of the Im8 protein is shown in SEQ ID NO.9;
  • the amino acid sequence of the Im9 protein is shown in SEQ ID NO.12.
  • the CL2 protein is mutated on the basis of the CE2 protein shown in SEQ ID NO.1;
  • the CL7 protein is mutated on the basis of the CE7 protein shown in SEQ ID NO.4;
  • the CL8 protein is mutated on the basis of the CE7 protein shown in SEQ ID NO.7;
  • the CL9 protein is mutated on the basis of the CE7 protein shown in SEQ ID NO.10.
  • the second object of the present invention is to provide the application of the protein complex in the preparation of artificial protein scaffolds.
  • the third object of the present invention is to provide a scaffold protein based on the protein complex, which includes: the cellulose binding module CBM3a, Im2 protein, Im7 protein, Im8 protein and Im9 protein connected in series through a linker ;
  • Each Im protein module on the scaffold protein can be connected with the corresponding CL protein to form an interaction pair, and the orderly assembly of the target protein on the scaffold protein can be realized by connecting the target protein with the CL protein.
  • the cellulose binding module CBM3a is derived from Clostridium thermocellum.
  • the linker includes: CBM3a and Im2 proteins are connected through Linker1 (TTTTTPTTASSTTTSTTSTSATSG), Im2 protein and Im7 protein are connected through Linker2 (NTLTIGGGNPGGGNPGGGTNPGT), Im7 protein and Im8 protein are connected through Linker3 (GASGSGTTTTTTTTSTTTGGTDP), Im8 Linker4 (SSWEGISTDDGIVHSVRISGNKTLS) was used to link the protein and Im9 protein.
  • Linker1 TTTTTPTTASSTTTSTTSTSATSG
  • NTLTIGGGNPGGGNPGGGTNPGT NTLTIGGGNPGGGNPGGGTNPGT
  • Im7 protein and Im8 protein are connected through Linker3 (GASGSGTTTTTTTTSTTTGGTDP)
  • Im8 Linker4 SSWEGISTDDGIVHSVRISGNKTLS was used to link the protein and Im9 protein.
  • the fourth object of the present invention is to provide the construction method of the scaffold protein, including:
  • Step 1 Obtain the gene fragments of CBM3a, Im2 protein, Im7 protein, Im8 protein and Im9 protein by PCR amplification method;
  • Step 2 using Overlapping PCR technology to sequentially connect the 5 gene fragments obtained in Step 1;
  • Step 3 constructing a recombinant vector with the gene connected in step 2, introducing it into recipient cells, and inducing expression to obtain the scaffold protein.
  • the fifth object of the present invention is to provide the application of the scaffold protein in the preparation of multi-enzyme complex system.
  • the sixth object of the present invention is to provide a multi-enzyme complex system based on the scaffold protein, which includes: 4 CL-tagged cellulase and the scaffold according to claim 5 Protein, 4 CL-tagged cellulases are connected with the corresponding Im protein module on the scaffold protein to form an interaction pair, that is, the multi-enzyme complex is obtained;
  • the four CL-tagged cellulases include: exoglucanase linked to CL2 protein, endoglucanase mutant linked to CL7 protein, endoglucanase linked to CL8 protein , and ⁇ -glucosidase linked to CL9 protein.
  • the exoglucanase is an exoglucanase Cel48S m3 obtained by DNA shuffling
  • the endoglucanase mutant is an endoglucanase mutant derived from Clostridium thermocellum Cel8A* (K276R, G283P, S329G, S375T)
  • the endoglucanase linked to CL8 protein is the endoglucanase GH5D derived from Caldicellulosiruptor bescii
  • the ⁇ -glucosidase is derived from Caldicellulosiruptor ⁇ -glucosidase CoGH1A of owensensis
  • the seventh object of the present invention is to provide the application of the multi-enzyme complex system in hydrolyzing cellulose substrates.
  • the present invention provides a protein complex based on colibactin E family DNase and its application in artificial protein scaffolds.
  • the carboxy-terminal DNase domains of enzymes CE2, CE7, CE8 and CE9 were protein engineered to obtain mutants CL2, CL7, CL8 and CL9 that lost DNase activity but still retained super high affinity with the corresponding immune protein Im protein , and constructed CL2/Im2, CL7/Im7, CL8/Im8 and CL9/Im9 protein interaction pairs.
  • CL2/Im2 CL7/Im7, CL8/Im8 and CL9/Im9 protein interaction pairs.
  • a platform for multi-enzyme assembly in a defined sequence provides a basis for constructing highly robust multi-enzyme molecular machines.
  • four kinds of cellulolytic enzymes are assembled on the artificial protein scaffold to form a multi-enzyme complex, which can hydrolyze the cellulose substrate more efficiently under high temperature environment.
  • the above protein interaction pairs can be used in any field involving protein-protein interaction.
  • Fig. 1 is the SDS-PAGE detection figure after 4 kinds of CL and Im protein purification in the embodiment 1 of the present invention
  • Fig. 2 uses Native-PAGE to detect the interaction between CL and Im protein (2-B) and the heat resistance of the interaction between CL and Im protein in Example 2 of the present invention (2-C);
  • Example 3 is a schematic diagram of the principle of scaffolding protein assembly of a multi-enzyme complex in Example 3 of the present invention
  • Fig. 4 is the SDS-PAGE detection result figure after the purification of the scaffold protein containing 1-4 Im modules in Example 3 of the present invention
  • Fig. 5 is the thermal stability detection result diagram of scaffold protein Scaf-CIQ in the embodiment 3 of the present invention.
  • Figure 6 is a diagram showing the detection results of the assembly of the multi-enzyme complex MEC-CIQ equipped with four cellulolytic enzymes in Example 4 of the present invention
  • Fig. 7 is a graph showing the synergistic detection results of 4 cellulolytic enzymes in the artificial multi-enzyme complex MEC-CIQ in Example 4 of the present invention.
  • CE2 protein as shown in SEQ ID NO.1
  • CL2 protein as shown in SEQ ID NO.2
  • Im2 protein as shown in SEQ ID NO.3;
  • CE7 protein as shown in SEQ ID NO.4
  • CL7 protein as shown in SEQ ID NO.5
  • Im7 protein as shown in SEQ ID NO.6;
  • CE8 protein as shown in SEQ ID NO.7; CL8 protein: as shown in SEQ ID NO.8; Im8 protein: as shown in SEQ ID NO.9;
  • CE9 protein as shown in SEQ ID NO.10; CL9 protein: as shown in SEQ ID NO.11; Im9 protein: as shown in SEQ ID NO.12.
  • the mutants and Im protein genes were handed over to Wuhan Jinkairui Company for gene synthesis, and the coding genes of the four CL and Im proteins were respectively constructed into the pET23a vector by conventional methods in the field.
  • the recombinant plasmid was transferred into Escherichia coli BL21(DE3), induced to express and purified, specifically: the expression strain was inoculated into LB liquid medium supplemented with 100 ⁇ g/mL ampicillin, and cultured at 37°C overnight; then the seed solution was mixed at a ratio of 1:100 Inoculate into TB medium at the ratio of OD 600 to 0.8-1.0, add IPTG with a final concentration of 1mM, induce at 18°C, 220rpm for 16-18h; collect the bacteria, and fully resuspend in TBS buffer (pH 7.4) Bacterial cells were crushed five times by a high-pressure cell disruptor, then centrifuged at 10,000 rpm for 30 minutes, and the supernatant was transferred to a
  • the pretreatment method is as follows: pipette 4mL nickel beads to a 60mL protein purification gravity column, and equilibrate the nickel beads with 3 times the volume of bacteria-breaking buffer. After transferring the supernatant to the gravity column, seal it with a parafilm, incubate on a silent mixer at 4°C for 1 h, then wash with 3 column volumes of TBS (containing 10 mM imidazole), and repeat 3 times. Then they were washed three times with TBS containing 30 mM, 50 mM and 200 mM successively, each time with 3 times the column volume, and the effluents were collected respectively. The collected solution after each gradient imidazole elution was detected by SDS-PAGE. The results are shown in Figure 1, and the results showed that all CL and Im proteins could be successfully expressed and purified. Recombinant protein samples were replaced by ultrafiltration and concentrated, then aliquoted and stored at -80°C.
  • an interaction pair can be formed, and the corresponding scaffold protein is constructed, as follows:
  • the scaffold protein Scaf-CIQ in the CIQ system is a cellulose-binding module CBM3a derived from Clostridium thermocellum (living environment: 60-90°C), and it is a fusion protein expressed in tandem with Im2, Im7, Im8, and Im9 through a linker, which is the scaffold Protein Scaf-CIQ.
  • CBM3a cellulose-binding module derived from Clostridium thermocellum (living environment: 60-90°C)
  • the presence of CBM3a endows the scaffold protein Scaf-CIQ with the ability to bind to cellulose matrices such as microcrystalline cellulose (Avicel), phosphoric acid swollen cellulose (PASC), and achieve the purpose of targeting cellulose matrices and immobilizing enzymes.
  • each Im module on the scaffold protein Scaf-CIQ can only be combined with the target protein such as enzyme that comprises corresponding CL-tag, thereby realizes 4 kinds of target proteins in the scaffold protein
  • the ordered assembly on Scaf-CIQ, the assembled complex is named MEC-CIQ (multienzyme complexes-CL-Im quartet).
  • the scaffold protein construction process is as follows:
  • the backbone of the vector is the pET23a vector that has been double-digested with NdeI and XhoI.
  • the forward primer of the CBM3a gene contains 15 nucleotides that are homologous to the flanking sequence of the NdeI site of the pET23a vector. According to the difference in the number of Im modules, The coding sequences of Im2, Im7, Im8 and Im9 each have a recombination sequence homologous to 15 bases flanking the XhoI site of the pET23a vector.
  • Linker1 amino acid sequence: TTTTTPTTASSTTTSTTSTSATSG, Inoue H, et al. Biotechnol Biofuels, 2015, 8, 77.
  • Linker2 amino acid sequence: NTLTIGGGNPGGGNPGGGTNPGT, Liu Y , et al. Ind Microbiol Biotechnol, 2015, 42, 1591-1599
  • Linker3 amino acid sequence: GASGSGTTTTTTTTSTTTGGTDP, Miao Y, et al.
  • Linker4 (amino acid sequence: SSWEGISTDDGIVHSVRISGNKTLS, Sakuraba H, et al. Biochim Biophys Acta, 2008, 1784, 563-571) is connected between Im8 protein and Im9 protein.
  • the scaffold protein of the Im2 module is named Scaf-CIM
  • the scaffold protein containing both Im2 and Im7 modules is named Scaf-CIB
  • the scaffold protein containing Im2, Im7 and Im8 is named Scaf-CIT, which also contains Im2
  • the scaffold protein of the four modules of , Im7, Im8 and Im9 was named Scaf-CIQ.
  • the recombinant vector was transformed into Escherichia coli BL21(DE3) for induced expression, and then purified by Ni-NTA method, and the purified protein was detected, the detection results are shown in Figure 4.
  • the results of SDS-PAGE showed that the scaffold proteins containing CBM3a and 1-4 Im modules could be purified by Ni-NTA to obtain higher purity proteins.
  • the carboxyl terminal of the scaffold protein Scaf-CIQ is Im9, and the amino terminal is CBM3a. It is known that Im9 specifically binds to CL9, and CBM3a can bind to phosphoric acid-swellable cellulose. Based on this, when exploring the thermal stability of Scaf-CIQ, phosphoric acid-swelled cellulose was used as the immobilization matrix, and sfGFP (CL9-sfGFP) fused with CL9-tag at the amino terminal was combined with Im9, which could be displayed on phosphoric acid-swelled cellulose. Green fluorescence. According to the intensity of green fluorescence, the integrity of Scaf-CIQ protein can be judged, that is, whether Scaf-CIQ after high temperature treatment retains the functions of CBM3a at the amino terminal and Im9 at the carboxyl terminal.
  • the scaffold protein Scaf-CIQ was incubated at 70°C, 75°C, 80°C, and 85°C for 3h and 6h, respectively, centrifuged at 12000rpm for 5min, and the supernatant was mixed with phosphoric acid-swelled cellulose and excess CL9-sfGFP was added. Incubate at 220 rpm on a shaker at 28°C for 1 h, then wash the phosphoric acid-swollen cellulose with 2M NaCl three times to remove free protein, resuspend the sample in 100 ⁇ L TBS buffer, and quantify the fluorescence intensity of CL9-sfGFP in each sample with a fluorescence spectrophotometer.
  • the fluorescence intensity measured by combining untreated Scaf-CIQ with CL9-sfGFP and phosphoric acid-swelled cellulose was 100%, and the sample without Scaf-CIQ added was used as a negative control.
  • the detection results of the relative fluorescence intensity are shown in Figure 5.
  • the results show that after culturing at 85°C for 3 hours, the binding ability of the scaffold protein Scaf-CIQ to cellulose and CL9-sfGFP is about 50%, and there is still A binding capacity of more than 30% indicates that the scaffold protein Scaf-CIQ of the present invention has high thermal stability, and therefore can be applied to the assembly and application of multi-enzyme complexes in high temperature environments.
  • the multi-enzyme complex assembly system MEC-CIQ is applied to the field of artificial cellulosomes.
  • the selected thermophilic cellulase genes include: (1) exoglucan Cel48S m3 obtained by DNA shuffling, (2) endoglucanase mutant Cel8A* (K276R, G283P, S329G, S375T), (3) endoglucanase GH5D derived from Caldicellulosiruptor bescii; (4) ⁇ -glucosidase CoGH1A derived from Caldicellulosiruptor owensensis (each cellulase refers to Smith MA, et al .Febs Journal.2012, 279, 4453-65; Anbar M., et al.
  • thermophilic cellulase genes are connected with the four CL protein genes to form CL-tagged thermophilic cellulase.
  • the specific operation process is as follows:
  • thermophilic cellulase genes were regarded as fragment A (A1, A2, A3 and A4), and the four CL protein genes As fragment B (B1, B2, B3 and B4), A and B are connected by a Pro/Thr-rich linker sequence (TTVTTPQTS).
  • TTVTTPQTS Pro/Thr-rich linker sequence
  • the reverse primer of B and the forward primer of A when the CL-tag is at the N-terminal of cellulase, the reverse primer of B and the forward primer of A contain an overlapping region of 15 bases in length, and the forward primer of B and the linear vector NcoI restriction site One end of the point contains 15 identical bases, and the reverse primer of A and the end of the linear vector XhoI restriction site contain 15 identical bases connected.
  • the reverse primer of A and the forward primer of B contain an overlapping region of 15 bases in length, and the forward primer of A and the end of the NcoI restriction site of the linear vector contain 15 bases. There are 15 identical bases connected between the reverse primer of B and the end of the XhoI restriction site of the linear vector.
  • the PCR product thus obtained had 15 bases identical to the end of the linear vector.
  • the PCR products A and B (such as A1 and B1 constituting genes A1-B1 or B1-A1) after the sequence of the fragments were determined were transformed into three fragments with the linearized vector under the mediation of T5 exonuclease, and passed through in Escherichia coli After homologous recombination, genes A, B and the linear vector are assembled into a complete circular plasmid containing the CL-tagged thermophilic cellulase gene.
  • All cellulase recombinant vectors were expressed by Escherichia coli BL21 (DE3) and purified by Ni-NTA.
  • the purified protein was combined with Scaf-CIQ to assemble into a heat-stable artificial cellulosome, so as to achieve a higher temperature in a high temperature environment.
  • the purpose of hydrolyzing the cellulose substrate, the specific operation is as follows:
  • the system was set at 500 ⁇ L, in which the final concentration of 4 CL-tagged cellulase was 3 ⁇ M, incubated with equimolar Scaf-CIQ at room temperature for 30 min, and carried out molecular sieve chromatography through the AKTA protein purification instrument.
  • the selected molecular sieve column The model is Superose 6Increase 10/300GL.
  • the assembly of the multi-enzyme complex was detected according to the peak value and SDS-PAGE, and the detection results are shown in FIG. 6 .
  • the molecular sieve result was a single peak, that is, with the participation of the scaffold protein Scaf-CIQ, CL2-Cel48S m3 , Cel8A*-CL7, GH5D-CL8, and CoGH1A-CL9 were successfully assembled together; and the SDS-PAGE results showed that the collection The peak contained the expected 5 proteins, and the molecular weights were as expected.
  • PASC was used as the cellulose substrate to conduct cellulase activity assays.
  • the measurement temperatures used were 65°C, 70°C, 75°C, and 80°C, and the reaction time was 3 hours.
  • the final concentration of each cellulase in 400 ⁇ L acetate buffer system containing 10 mM CaCl 2 was 1 ⁇ M, and the amount of PASC was 2.5%.
  • the completed reaction system was centrifuged at 12,000 rpm for 1 min, and 120 ⁇ L of supernatant was mixed with 180 ⁇ L of DNS solution. After being treated in a boiling water bath for 10 min, it was immediately cooled in an ice-water mixture. Centrifuge at 6000 rpm for 1 min, take 250 ⁇ L of the reaction solution and measure the absorbance at OD 540 .
  • Substitute the measured value into the measured glucose standard curve calculate the glucose content generated by each group of reactions, and set up three independent parallel groups for each group of experiments, and use the free four kinds of cellulose that have not been assembled with multi-enzyme complexes hydrolase as a control.
  • MEC-CIQ system had the largest increase in reducing sugar production after reacting at 70°C for 3 hours, which was 160% of the free enzyme system. Based on this, the MEC-CIQ system constructed in the present invention can be successfully applied to the assembly of multi-enzyme complexes, and exhibits excellent performance in a high-temperature environment.

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Abstract

本发明公开了基于大肠杆菌素E家族DNA酶的蛋白质复合物及其在人工蛋白支架中的应用，所述蛋白质复合物中包括：CL2蛋白与Im2蛋白形成的相互作用对，或CL7蛋白与Im7蛋白形成的相互作用对，或CL8蛋白与Im8蛋白形成的相互作用对，或CL9蛋白与Im9蛋白形成的相互作用对中的任意一种或多种。本发明通过对CE家族的DNA酶CE2、CE7、CE8和CE9的羧基端DNase结构域进行蛋白质工程改造，获得丧失DNA酶活性，但仍保留有与对应的免疫蛋白Im具有超高亲和力的突变体，并构建得到了CL2/Im2，CL7/Im7，CL8/Im8和CL9/Im9蛋白质相互作用对。研究发现这些蛋白质相互作用对具有耐热性，高亲和力，高特异性，分子量小，组装速度快等性质，基于此构建了人工蛋白质支架，为人工多酶复合物的构建搭建了新型平台。

Description

基于大肠杆菌素E家族DNA酶的蛋白质复合物及其在人工蛋白支架中的应用 技术领域
本发明属于合成生物学及纳米生物技术领域，具体涉及基于大肠杆菌素E家族DNA酶的蛋白质复合物及其在人工蛋白支架中的应用。
背景技术
高亲和力的蛋白质相互作用对在许多领域都有广泛的应用，如免疫共沉淀、蛋白质pull-down、酵母双杂交和酶的固定化等，其中利用蛋白质相互作用对自组装形成的多酶复合物体系因具备出色的协同催化能力而备受关注。对于“一锅法”合成反应而言，利用多个有着正交性的蛋白质相互作用对实现多个酶分子按照特定的顺序进行组装，以人工多酶复合物的形式进行级联催化反应，有利于底物通道的形成，可以防止中间产物的扩散，促进中间产物及时转化，从而避免某些有毒中间体对酶活性的干扰，提高反应效率。目前，蛋白质-蛋白质的相互作用主要通过来源于纤维小体的基本组装元件Cohesin-Dockerin以及人工设计的SpyCatcher/SpyTag、SnoopCatcher/SnoopTag和RIAD/RIDD等蛋白质相互作用对实现。然而现有的蛋白质相互作用对在实际应用时存在诸多问题。首先，强相互作用对的数量有限，目前常用的相互作用对的亲和力都不强，在构建较为复杂的多酶复合物时，结构不稳定，容易坍塌。其次，对于某些放热反应，如纤维素的降解，需要热稳定的蛋白质相互作用对结合热稳定酶来构建多酶复合物，目前此类蛋白质相互作用对更是少之又少。此外，大多数蛋白质相互作用对都需要在高盐离子条件下发生相互作用，如Cohesin-Dockerin的作用受钙离子调控，限制了它们在体内的应用。因此，针对以上问题挖掘具有优良性质的新蛋白质相互作用对具有重要意义。
大肠杆菌产生的大肠杆菌素是一种细胞毒素，可以杀死亲缘关系比较近的杆菌。大肠杆菌素E家族的DNA酶包括CE2、CE7、CE8和CE9，是一类结构高度相似的非专一性核酸内切酶。在宿主细胞中，CE蛋白与各自对应的免疫蛋 白Im2、Im7、Im8和Im9结合，抑制CE蛋白与DNA的结合，从而消除它们对于宿主的毒性。CE蛋白和Im蛋白的结合是目前自然界已知最强的蛋白质相互作用之一，K d达到10 -14～10 -17。Im与CE的结合为“双重识别机制”，即Im蛋白质首先通过保守元件(螺旋Ⅲ)与CE核酸酶形成序列无关的复合物；随后Im蛋白质上含有特异性残基的螺旋Ⅱ与对应的CE核酸酶结合，稳定正交复合物(Li W.,et al,Biochemistry.1998,37,11771-11779；Keeble AH,Kleanthous C.,J Mol Biol.2005,352,656-671)。
发明内容
本发明的目的在于提供基于大肠杆菌素E家族DNA酶的蛋白质复合物及其在人工蛋白支架中的应用，本发明通过对CE家族的DNA酶CE2、CE7、CE8和CE9的羧基端DNase结构域进行蛋白质工程改造，获得丧失DNA酶活性，但仍保留与对应Im蛋白的超高亲和力的突变体，并构建得到了CL2/Im2，CL7/Im7，CL8/Im8和CL9/Im9蛋白质相互作用对。研究发现这些蛋白质相互作用对具有耐热性，高亲和力，高特异性，分子量小，组装速度快等性质，基于此构建了人工蛋白质支架，为人工多酶复合物的构建搭建了新型平台。
本发明的目的之一在于提供一种基于大肠杆菌素E家族DNA酶的蛋白质复合物，所述蛋白质复合物中包括：CL2蛋白与Im2蛋白形成的相互作用对，或CL7蛋白与Im7蛋白形成的相互作用对，或CL8蛋白与Im8蛋白形成的相互作用对，或CL9蛋白与Im9蛋白形成的相互作用对中的任意一种或多种；其中，
所述CL2蛋白的氨基酸序列如SEQ ID NO.2所示；
所述CL7蛋白的氨基酸序列如SEQ ID NO.5所示；
所述CL8蛋白的氨基酸序列如SEQ ID NO.8所示的；
所述CL9蛋白的氨基酸序列如SEQ ID NO.11所示。
进一步地，
所述Im2蛋白的氨基酸序列如SEQ ID NO.3所示；
所述Im7蛋白的氨基酸序列如SEQ ID NO.6所示；
所述Im8蛋白的氨基酸序列如SEQ ID NO.9所示；
所述Im9蛋白的氨基酸序列如SEQ ID NO.12所示。
进一步地，
所述CL2蛋白是在如SEQ ID NO.1所示的CE2蛋白基础上突变得到的；
所述CL7蛋白是在如SEQ ID NO.4所示的CE7蛋白基础上突变得到的；
所述CL8蛋白是在如SEQ ID NO.7所示的CE7蛋白基础上突变得到的；
所述CL9蛋白是在如SEQ ID NO.10所示的CE7蛋白基础上突变得到的。
本发明的目的之二在于提供了所述蛋白质复合物在制备人工蛋白质支架中的应用。
本发明的目的之三在于提供了一种基于所述蛋白质复合物的支架蛋白，所述支架蛋白中包括：通过linker依次串联的纤维素结合模块CBM3a，Im2蛋白，Im7蛋白，Im8蛋白以及Im9蛋白；
所述支架蛋白上的各Im蛋白模块可与相应的CL蛋白连接形成相互作用对，通过将目的蛋白与CL蛋白连接，实现目的蛋白在支架蛋白上的有序装配。
进一步地，所述纤维素结合模块CBM3a来源于热纤梭菌Clostridium thermocellum。
进一步地，所述linker包括：CBM3a与Im2蛋白之间通过Linker1(TTTTTPTTASSTTSTTSTSATSG)连接，Im2蛋白与Im7蛋白之间通过Linker2(NTLTIGGGNPGGGNPGGGTNPGT)连接，Im7蛋白和Im8蛋白之间通过Linker3(GASGSGTTTTTTTTSTTTGGTDP)连接，Im8蛋白和Im9蛋白之间通过Linker4(SSWEGISTDDGIVHSVRISGNKTLS)连接。
本发明的目的之四在于提供了所述支架蛋白的构建方法，包括：
步骤一、采用PCR扩增的方法分别得到CBM3a，Im2蛋白，Im7蛋白，Im8蛋白以及Im9蛋白的基因片段；
步骤二、采用Overlapping PCR技术将步骤一得到的5段基因片段依次连接；
步骤三、将步骤二连接好的基因进行重组载体构建，并导入受体细胞，诱导表达，得到所述支架蛋白。
本发明的目的之五在于提供了所述支架蛋白在制备多酶复合物体系中的应用。
本发明的目的之六在于提供了一种基于所述支架蛋白的多酶复合物体系，所述多酶复合物体系中包括：4个CL-tagged纤维素酶以及如权利要求5所述的支架蛋白，4个CL-tagged纤维素酶与支架蛋白上对应的Im蛋白模块连接形成相互作用对，即得到所述多酶复合物；
其中所述4个CL-tagged纤维素酶包括：与CL2蛋白连接的外切葡聚糖酶，与CL7蛋白连接的内切葡聚糖酶突变体，与CL8蛋白连接的内切葡聚糖酶，以及与CL9蛋白连接的β-葡萄糖苷酶。
进一步地，所述外切葡聚糖酶为通过DNA shuffling得到的外切葡聚糖酶Cel48S m3，所述内切葡聚糖酶突变体为来源于Clostridium thermocellum的内切葡聚糖酶突变体Cel8A*(K276R,G283P,S329G,S375T)，所述与CL8蛋白连接的内切葡聚糖酶为来源于Caldicellulosiruptor bescii的内切葡聚糖酶GH5D，所述β-葡萄糖苷酶为来源于Caldicellulosiruptor owensensis的β-葡萄糖苷酶CoGH1A
本发明的目的之七在于提供了所述多酶复合物体系在水解纤维素底物中的应用。
与现有技术相比，本发明的有益效果是：本发明提供了一种基于大肠杆菌素E家族DNA酶的蛋白质复合物及其在人工蛋白支架中的应用，本发明通过对CE家族的DNA酶CE2、CE7、CE8和CE9的羧基端DNase结构域进行蛋白质工程改造，获得丧失DNA酶活性，但仍保留有与对应的免疫蛋白Im蛋白具有超高亲和力的突变体CL2、CL7、CL8和CL9，并构建得到了CL2/Im2，CL7/Im7，CL8/Im8和CL9/Im9蛋白质相互作用对。研究发现上述CL/Im蛋白质相互作用 对工具箱具有高特异性、超高亲和力和高热稳定性、结构统一等特性，因此基于该CL/Im工具箱可构建稳定、耐热蛋白质支架，并能够按照设定的顺序进行多酶组装的平台，为构建具备高鲁棒性的多酶分子机器提供了基础。本发明中将4种纤维素水解酶组装到该人工蛋白质支架上形成多酶复合物，在高温环境下更高效水解纤维素底物。同时，在任何涉及到蛋白质-蛋白质相互作用的领域都能够使用以上蛋白质相互作用对。
附图说明
图1为本发明实施例1中4种CL与Im蛋白纯化后的SDS-PAGE检测图；
图2为本发明实施例2中采用Native-PAGE检测CL和Im蛋白质的相互作用(2-B)以及CL和Im蛋白质之间相互作用的耐热性(2-C)；
图3为本发明实施例3中支架蛋白组装多酶复合物的原理示意图；
图4为本发明实施例3中含有1-4个Im模块的支架蛋白纯化后的SDS-PAGE检测结果图；
图5为本发明实施例3中支架蛋白Scaf-CIQ的热稳定性检测结果图；
图6为本发明实施例4中装配有4种纤维素水解酶的多酶复合物MEC-CIQ的组装情况检测结果图；
图7为本发明实施例4中人工多酶复合物MEC-CIQ中4中纤维素水解酶的协同作用检测结果图。
具体实施方式
下面将结合本发明中的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1 CL蛋白与Im蛋白的表达与纯化
1、通过与CL7蛋白质进行氨基酸序列比对，基于4种蛋白质序列的高度相 似性，将CE家族的DNA酶CE2、CE7、CE8和CE9的羧基端DNase结构域进行蛋白质工程改造，获得丧失DNA酶活性，但仍保留有与对应的免疫蛋白Im蛋白具有超高亲和力的突变体CL2、CL7、CL8和CL9，其中四组CL-Im及4种野生型CE蛋白质序列为：
(1)CE2蛋白：如SEQ ID NO.1所示；CL2蛋白：如SEQ ID NO.2所示；Im2蛋白：如SEQ ID NO.3所示；
(2)CE7蛋白：如SEQ ID NO.4所示；CL7蛋白：如SEQ ID NO.5所示；Im7蛋白：如SEQ ID NO.6所示；
(3)CE8蛋白：如SEQ ID NO.7所示；CL8蛋白：如SEQ ID NO.8所示；Im8蛋白：如SEQ ID NO.9所示；
(4)CE9蛋白：如SEQ ID NO.10所示；CL9蛋白：如SEQ ID NO.11所示；Im9蛋白：如SEQ ID NO.12所示。
2、各突变体及Im蛋白基因交由武汉金开瑞公司进行基因合成，将4种CL和Im蛋白的编码基因分别采用本领域常规方法构建至pET23a载体中。重组质粒转入大肠杆菌BL21(DE3)，诱导表达并纯化，具体为：表达菌株接种到添加100μg/mL氨苄青霉素的LB液体培养基中，37℃培养过夜；其后将种子液按1:100的比例接种到TB培养基中，待OD 600值达到0.8～1.0时，加入终浓度为1mM的IPTG，18℃，220rpm诱导16～18h；收集菌体，用TBS buffer(pH 7.4)充分重悬菌体，高压细胞破碎仪重复破碎5遍，随后用10000rpm，30min离心，将上清转移到预处理好的重力柱中。
预处理方法如下：吸取4mL镍珠至60mL蛋白纯化重力柱，用3倍体积破菌buffer平衡镍珠。将上清转移至重力柱后，封口膜封口，于静音混合器上4℃孵育1h，然后用3倍柱体积的TBS(含10mM咪唑)洗涤，重复3次。随后依次用含30mM、50mM、200mM的TBS洗涤各3次，每次3倍柱体积，分别收集流出液。将各梯度咪唑洗脱后的收集液用SDS-PAGE检测。结果如图1 所示，结果显示，所有CL与Im蛋白质均能成功表达并纯化。重组蛋白样品经超滤换液并浓缩后分装，于-80℃冷冻保存。
实施例2 Native-PAGE
1、Native-PAGE非变性丙烯酰胺凝胶制备
对于CL-Im蛋白质相互作用对的结合特异性验证，采用30％丙烯酰胺(37.5:1)、甘油电泳体系，具体配方如表1所示。
表1 Native-PAGE非变性丙烯酰胺凝胶配方
Figure PCTCN2022102567-appb-000001
2、Native-PAGE
为验证CL蛋白与Im蛋白结合的特异性，本实施例将4种CL蛋白质和4种Im蛋白质分别混合，得到16种组合，室温孵育30min后进行Native-PAGE，上层胶时电压为120V，下层胶电压为180V，当溴酚蓝指示带泳动到胶底部时结束电泳，然后进行染色。电泳检测结果如图2-B所示，电泳结果表明CL与Im之间的相互作用具有良好的特异性。
进一步验证CL与Im蛋白质结合的热稳定性，具体为：将16种组合的CL-Im蛋白质混合体系，室温孵育30min后，将其分别在100℃水浴加热1h，对高温处理后的蛋白质混合样品进行Native-PAGE，检测结果如图2-C所示。结果表明Im和CL蛋白在高温处理后发生微弱结构变化，导致活性胶蛋白质条带模糊，但彼此正交的4组Im-CL形成的复合物在高温环境下均没有发生明显变化，由 此可知Im与CL的相互作用具有良好的热稳定性。
实施例3支架蛋白Scaf-CIQ的构建及热稳定性验证
1、本实施例基于上述CL与Im蛋白质结合可形成相互作用对，构建相应的支架蛋白，具体如下：
CIQ系统中的支架蛋白Scaf-CIQ为来源于Clostridium thermocellum(生存环境为60～90℃)的纤维素结合模块CBM3a，通过linker与Im2、Im7、Im8和Im9串联表达出的融合蛋白，即为支架蛋白Scaf-CIQ。CBM3a的存在赋予支架蛋白Scaf-CIQ与微晶纤维素(Avicel)、磷酸膨胀纤维素(PASC)等纤维素基质结合的能力，达到靶向纤维素基质和固定化酶的目的。所述支架蛋白Scaf-CIQ的原理如图3所示，支架蛋白Scaf-CIQ上的各Im模块只能与包含相应CL-tag的目的蛋白如酶进行结合，从而实现4种目的蛋白在支架蛋白Scaf-CIQ上的有序装配，组装而成的复合物被命名为MEC-CIQ(multienzyme complexes-CL-Im quartet)。
所述支架蛋白构建过程如下：
通过PCR扩增出四种Im蛋白及CBM3a的对应基因片段，采用Overlapping PCR技术将其整合，得到含有1-4个Im模块的基因序列，并进行重组载体构建，具体为：所述支架蛋白选用的载体骨架为经过NdeⅠ和XhoⅠ双酶切的pET23a载体，设计引物时，CBM3a基因的正向引物含有15个与pET23a载体NdeⅠ位点侧翼序列同源的核苷酸，根据Im模块数量的不同，Im2、Im7、Im8和Im9编码序列分别带有与pET23a载体XhoⅠ位点侧翼15个碱基同源的重组序列。
其中CBM3a与Im2蛋白之间通过Linker1(氨基酸序列：TTTTTPTTASSTTSTTSTSATSG，Inoue H,et al.Biotechnol Biofuels,2015,8,77.)连接，Im2蛋白与Im7蛋白之间通过Linker2(氨基酸序列：NTLTIGGGNPGGGNPGGGTNPGT,Liu Y,et al.Ind Microbiol Biotechnol,2015,42,1591-1599)连接，Im7蛋白和Im8蛋白之间通过Linker3(氨基酸序列： GASGSGTTTTTTTTSTTTGGTDP,Miao Y,et al.AMB Express,2018,8,44)连接，Im8蛋白和Im9蛋白之间通过Linker4(氨基酸序列：SSWEGISTDDGIVHSVRISGNKTLS,Sakuraba H,et al.Biochim Biophys Acta,2008,1784,563-571)连接。
PCR扩增出含不同Linker序列的四种Im蛋白质及CBM3a的基因片段，将CBM3a、Im2(Im2+Im7、Im2+Im7+Im8，Im2+Im7+Im8+Im9)和线性化的pET23a线性载体按摩尔比3:1混合，5μL体系中加入0.5U的T5核酸外切酶及对应的NEB Buffer4，加入大肠杆菌DH5α感受态后，冰水混合浴上静置5min，基因的同源臂区域将产生3’端突出的黏性末端，基因片段转入感受态细胞后，在大肠杆菌体内经过同源重组，将得到含有1-4个Im模块与CBM3a完整基因序列的环状载体，其中仅包含一个Im2模块的支架蛋白命名为Scaf-CIM，同时包含Im2和Im7两个模块的支架蛋白命名为Scaf-CIB，同时包含Im2、Im7和Im8三个模块的支架蛋白命名为Scaf-CIT，同时包含Im2、Im7、Im8和Im9四个模块的支架蛋白命名为Scaf-CIQ。
重组载体转入大肠杆菌BL21(DE3)进行诱导表达，继而通过Ni-NTA方法进行纯化，并对纯化蛋白进行检测，检测结果如图4所示。SDS-PAGE结果表明，含CBM3a和1-4个Im模块的支架蛋白均能通过Ni-NTA纯化方法得到较高纯度的蛋白质。
2、为探究所述支架蛋白Scaf-CIQ的热稳定性，所采用的方法如下：
所述支架蛋白Scaf-CIQ的羧基端为Im9，氨基端为CBM3a，已知Im9与CL9特异性结合，CBM3a可与磷酸膨胀纤维素结合。基于此，探究Scaf-CIQ热稳定性时，采用磷酸膨胀纤维素为固定化基质，通过氨基端融合有CL9-tag的sfGFP(CL9-sfGFP)与Im9的结合，可在磷酸膨胀纤维素上呈现绿色荧光。根据绿色荧光的强弱，可判断出Scaf-CIQ蛋白质的完整性，即高温处理后的Scaf-CIQ是否保留有氨基端的CBM3a与羧基端的Im9功能。
本实施例中将支架蛋白Scaf-CIQ于70℃、75℃、80℃和85℃条件下分别孵育3h和6h，12000rpm离心5min，取上清与磷酸膨胀纤维素混合并加入过量CL9-sfGFP，28℃摇床220rpm孵育1h，然后用2M NaCl洗涤磷酸膨胀纤维素3次，去除游离蛋白质，用100μL TBS buffer重悬样品，采用荧光分光光度计对各样品中的CL9-sfGFP进行荧光强度定量。以未处理的Scaf-CIQ与CL9-sfGFP、磷酸膨胀纤维素结合测得的荧光强度为100％，以未添加Scaf-CIQ的样品作为负对照。相对荧光强度的检测结果如图5所示，结果显示，在85℃下培养3h后，所述支架蛋白Scaf-CIQ对纤维素和CL9-sfGFP的结合能力约为50％，在6h后仍有30％以上的结合能力，即说明本发明所述的支架蛋白Scaf-CIQ具有较高的热稳定性，因此可应用于高温环境下的多酶复合物组装和应用。
实施例4多酶复合物MEC-CIQ的组装及应用
1、含4种纤维素水解酶的多酶复合物MEC-CIQ的组装情况
本实施例将多酶复合物组装系统MEC-CIQ应用于人工纤维小体领域。具体为：选用的嗜热纤维素酶基因包括：(1)通过DNA shuffling得到的外切葡聚糖Cel48S m3、(2)来源于Clostridium thermocellum的内切葡聚糖酶突变体Cel8A*(K276R,G283P,S329G,S375T)、(3)来源于Caldicellulosiruptor bescii的内切葡聚糖酶GH5D；(4)来源于Caldicellulosiruptor owensensis的β-葡萄糖苷酶CoGH1A(各纤维素酶分别参考文献Smith M.A.,et al.Febs Journal.2012,279,4453-65；Anbar M.,et al.Appl Environ Microbiol.2012,78,3458；Dvortsov IA,et al.,Int J Biol Macromol,2018,107,305-11；Peng X.,et al.Biotechnol Biofuels,2016,9,98.)。
采用将上述四种嗜热纤维素酶基因别与四种CL蛋白基因连接，形成CL-tagged嗜热纤维素酶，具体操作过程为：
以经过NcoⅠ和XhoⅠ双酶切并琼脂糖凝胶回收后的pET28a为载体骨架，将上述四种嗜热纤维素酶基因视为片段A(A1、A2、A3和A4)，四种CL蛋白 基因视为片段B(B1、B2、B3和B4)，A和B之间通过富含Pro/Thr的linker序列(TTVTTPQTS)连接。设计引物时，CL-tag在纤维素酶的N端情况下，B的反向引物和A的正向引物含有15个碱基长度的重叠区，B的正向引物和线性载体NcoⅠ酶切位点一端含有15个相同的碱基，A的反向引物和线性载体XhoⅠ酶切位点一端含有15个相同的碱基连接。CL-tag在纤维素酶的C端情况下，A的反向引物和B的正向引物含有15个碱基长度的重叠区，A的正向引物和线性载体NcoⅠ酶切位点一端含有15个相同的碱基，B的反向引物和线性载体XhoⅠ酶切位点一端含有15个相同的碱基连接。如此得到的PCR产物和线性载体的末端有15个相同的碱基。将片段顺序确定后的PCR产物A和B(如A1和B1组成基因A1-B1或B1-A1)与线性化载体在T5核酸外切酶的介导下进行三片段转化，在大肠杆菌体内经过同源重组后，基因A、B和线性载体组装成完整的环状质粒，含有CL-tagged嗜热纤维素酶基因。
所有纤维素酶的重组载体均通过大肠杆菌BL21(DE3)表达，并通过Ni-NTA方式纯化，纯化后的蛋白质与Scaf-CIQ搭配组装成热稳定人工纤维小体，从而达到在高温环境下更好水解纤维素底物的目的，具体操作如下：
本实施例中将体系定为500μL，其中4个CL-tagged纤维素酶终浓度为3μM，室温条件下与等摩尔Scaf-CIQ孵育30min，通过AKTA蛋白纯化仪进行分子筛层析，选用的分子筛柱型号为Superose 6Increase 10/300GL。以TBS buffer为纯化buffer，流速为0.5mL/min。根据峰值和SDS-PAGE检测多酶复合物的组装，检测结果如图6所示。结果显示，分子筛结果为单一峰，即在支架蛋白Scaf-CIQ的参与下，CL2-Cel48S m3、Cel8A*-CL7、GH5D-CL8、CoGH1A-CL9成功组装在一起；并且SDS-PAGE结果表明该收集峰中含有预期的5种蛋白质，且分子量与预期一致。
2、多酶复合物MEC-CIQ中4种纤维素水解酶的协同作用
为验证组装成功的人工纤维小体，即包含4种纤维水解酶的多酶复合物 MEC-CIQ的性能，采用PASC为纤维素底物，进行纤维素酶活性测定。
采用的测定温度为65℃、70℃、75℃、80℃，反应时间为3h。在含有10mM CaCl 2的400μL醋酸缓冲液体系中各种纤维素酶的终浓度为1μM，PASC的量为2.5％。将反应完毕的体系12000rpm离心1min，取120μL上清与180μL DNS溶液混合，沸水浴处理10min后立即置于冰水混合物中冷却。6000rpm离心1min，取250μL反应液在OD 540处测定吸光度。将测得的数值代入至测定的葡萄糖标准曲线中，计算出每组反应生成的葡萄糖含量，每组实验均设置三组独立平行，并以未进行多酶复合物组装的游离的4种纤维素水解酶作为对照。
检测结果如图7所示，结果显示以PASC为底物时，每组实验中均能体现出多酶协同作用优势。其中，MEC-CIQ体系在70℃反应3h后的还原糖产量提升幅度最大，为游离酶体系的160％。基于此，本发明构建的MEC-CIQ系统可成功应用于多酶复合物组装，且在高温环境下表现出卓越的性能。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种基于大肠杆菌素E家族DNA酶的蛋白质复合物，其特征在于，所述蛋白质复合物中包括：CL2蛋白与Im2蛋白形成的相互作用对，或CL7蛋白与Im7蛋白形成的相互作用对，或CL8蛋白与Im8蛋白形成的相互作用对，或CL9蛋白与Im9蛋白形成的相互作用对中的任意一种或多种；其中，
    所述CL2蛋白的氨基酸序列如SEQ ID NO.2所示；
    所述CL7蛋白的氨基酸序列如SEQ ID NO.5所示；
    所述CL8蛋白的氨基酸序列如SEQ ID NO.8所示的；
    所述CL9蛋白的氨基酸序列如SEQ ID NO.11所示。
  2. 根据权利要求1所述的蛋白质复合物，其特征在于，
    所述Im2蛋白的氨基酸序列如SEQ ID NO.3所示；
    所述Im7蛋白的氨基酸序列如SEQ ID NO.6所示；
    所述Im8蛋白的氨基酸序列如SEQ ID NO.9所示；
    所述Im9蛋白的氨基酸序列如SEQ ID NO.12所示。
  3. 根据权利要求1所述的蛋白质复合物，其特征在于，
    所述CL2蛋白是在如SEQ ID NO.1所示的CE2蛋白基础上突变得到的；
    所述CL7蛋白是在如SEQ ID NO.4所示的CE7蛋白基础上突变得到的；
    所述CL8蛋白是在如SEQ ID NO.7所示的CE7蛋白基础上突变得到的；
    所述CL9蛋白是在如SEQ ID NO.10所示的CE7蛋白基础上突变得到的。
  4. 如权利要求1-3任一项所述的蛋白质复合物在制备人工蛋白质支架中的应用。
  5. 一种基于权利要求1-3任一项所述的蛋白质复合物的支架蛋白，其特征在于，所述支架蛋白中包括：通过linker依次串联的纤维素结合模块CBM3a，Im2蛋白，Im7蛋白，Im8蛋白以及Im9蛋白；
    所述支架蛋白上的各Im蛋白模块可与相应的CL蛋白连接形成相互作用对，通过将目的蛋白与CL蛋白连接，实现目的蛋白在支架蛋白上的有序装配。
  6. 根据权利要求5所述的支架蛋白，其特征在于，所述纤维素结合模块CBM3a来源于热纤梭菌Clostridium thermocellum。
  7. 如权利要求5所述的支架蛋白的构建方法，其特征在于，所述方法包括：
    步骤一、采用PCR扩增的方法分别得到CBM3a，Im2蛋白，Im7蛋白，Im8蛋白以及Im9蛋白的基因片段；
    步骤二、采用Overlapping PCR技术将步骤一得到的5段基因片段依次连接；
    步骤三、将步骤二连接好的基因进行重组载体构建，并导入受体细胞，诱导表达，得到所述支架蛋白。
  8. 如权利要求5所述的支架蛋白在制备多酶复合物体系中的应用。
  9. 一种基于权利要求5所述的支架蛋白的多酶复合物体系，其特征在于，所述多酶复合物体系中包括：4个CL-tagged纤维素酶以及如权利要求5所述的支架蛋白，4个CL-tagged纤维素酶与支架蛋白上对应的Im蛋白模块连接形成相互作用对，即得到所述多酶复合物；
    其中所述4个CL-tagged纤维素酶包括：与CL2蛋白连接的外切葡聚糖酶，与CL7蛋白连接的内切葡聚糖酶突变体，与CL8蛋白连接的内切葡聚糖酶，以及与CL9蛋白连接的β-葡萄糖苷酶。
  10. 如权利要求9所述的多酶复合物体系在水解纤维素底物中的应用。
PCT/CN2022/102567 2021-10-27 2022-06-30 基于大肠杆菌素e家族dna酶的蛋白质复合物及其在人工蛋白支架中的应用 WO2023071267A1 (zh)

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