CN111850007B

CN111850007B - Cellulosobody docking protein combination mutant 36864 applicable to low calcium ion concentration and application

Info

Publication number: CN111850007B
Application number: CN202010730981.0A
Authority: CN
Inventors: 汪俊卿; 王大涛; 李楠; 范翰; 王瑞明; 李丕武; 姜彦君; 王子睿; 杨翠平; 薛乐; 张晨曦
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2022-03-29
Anticipated expiration: 2040-07-27
Also published as: CN111850007A

Abstract

A small-fiber docking protein combination mutant 36864 suitable for low calcium ion concentration is a small-fiber docking protein DocA, the nucleotide sequence is shown as SEQ ID No.3, and fixed-point mutation is carried out to obtain a docking protein combination mutant 36864 suitable for low calcium ion concentration, and the nucleotide sequence is shown as SEQ ID No. 1; the dockerin mutant is suitable for low calcium ion concentration of 10^‑7M‑10^‑4The condition of M interacts with mucin; further suitable for low calcium ion concentration of 10^‑7M‑10^‑6The condition of M interacts with mucin; is suitable for interaction with mucin in an intracellular environment; solves the problem that the existing fiber body element can not be self-assembled under the condition of low calcium ion concentration.

Description

Cellulosobody docking protein combination mutant 36864 applicable to low calcium ion concentration and application

Technical Field

The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a cellosome docking protein combination mutant 36864 suitable for low calcium ion concentration and application thereof.

Background

The cellulosome is a natural extracellular multienzyme self-assembly system which can organize and coordinate a plurality of enzyme components to efficiently catalyze and degrade lignocellulose in a synergetic way, the cellulosome is not only a supermolecule extracellular protein complex formed by the interaction between foot rest protein, cellulase, hemicellulase and other enzymes through an assembly module, namely fibronectin and docking protein, but also a nanometer machine which is composed of a plurality of non-catalytic and catalytic subunits and has a complex structure, the existence of the nanometer machine can greatly improve the degradation efficiency of cellulose, and researches show that the efficiency of the cellulosome for hydrolyzing cellulose is 6 times of that of free enzyme, and the cellulosome can be attached to the surface of bacterial cells and can be independently released as a cellfree cellulosome. The fibroid consists essentially of two parts: dockerin (Doc) containing enzymes or other accessory proteins and fibronectin (cohesin, Coh) containing structural proteins.

The research of the prior art finds that a sequence D rich in hydrophilic amino acid exists in a cellulosome element Doc¹ D/N ³D/N ⁵ D/N⁹ D¹²And the affinity of Doc and Coh from different producer species for each other can be altered by mutating the sequence between S688-T689 and S720-T721 therein, see the documents [ Page' S. PROTECTINS: Structure, Function, and Genetics,1997,29: 517-527; MECHANY PROTEINS Structure, Function, and Genetics 2000,39: 170-](ii) a The binding between Coh and Doc in the prior art requires large amounts of calcium ions (concentrations of 0.5mM to 2mM), see literature [ bulb, Journal of Biological Chemistry,2018,293 (11); xu, Biotechnol Biofuels,2013, 6(1):126](ii) a Even higher calcium ion concentrations (2 mM-10 mM), see literature [ MECHANY. PROTECTINS: Structure, Function, and Genetics,2000,39: 170-]。

Due to metabolic requirements, the calcium ion concentration in the cells of microorganisms at rest is generally only 10^-7M～10^-6M, much lower than the extracellular concentration (1mM), see the literature [ Van Qian, Chinese Tropical medicine, 2014,14(11):1309-1313]Therefore, the cellulosome can only act at high calcium ion concentration (. gtoreq.1 mM) in the prior art, which fails to provide a solution that can be at 10 ≥ mM^-7M ～10^-6The cellosome element that can be efficiently assembled under M condition cannot be provided in a wide range of calcium ion environment (calcium ion concentration 10)^-7M-2 mM), and further fails to provide a cellulosome element capable of functioning in biological cells (calcium ion concentration 10 or less)^-6M) assembled cellosome elements.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a small-body docking protein combined mutant 36864 suitable for low calcium ion concentration and application thereof.

The invention provides a cellosome dockerin mutant suitable for low calcium ion concentration and application thereof, solves the problem that the existing cellosome element cannot be self-assembled under low calcium ion concentration (such as in cells), and provides a mutant suitable for a wide-range calcium ion environment (calcium ion concentration 10)^-7M～2×10^-3M) a working cellulosome dockerin mutant.

The technical scheme of the invention

A small-fiber docking protein combined mutant 36864 suitable for low calcium ion concentration is a small-fiber docking protein DocA, the nucleotide sequence is shown as SEQ ID No.3, and site-directed mutagenesis is performed to obtain the docking protein combined mutant 36864 suitable for low calcium ion concentration, and the nucleotide sequence is shown as SEQ ID No. 1.

A small-fiber docking protein combined mutant 36864 suitable for low calcium ion concentration is a small-fiber docking protein DocA, the amino acid sequence is shown as SEQ ID No.4, and site-directed mutagenesis is performed to obtain the docking protein combined mutant 36864 suitable for low calcium ion concentration, and the amino acid sequence is shown as SEQ ID No. 2.

The DocA is derived from Clostridium thermocellum (Clostridia thermocellum) (GenBank:2CCL _ B), the nucleotide sequence of the DocA is shown as SEQ ID NO.3, and the amino acid sequence of the DocA is shown as SEQ ID NO. 4.

D in the above amino acid sequence₆VNGDGTINSTD₁₇Mutation to D₆VDGSGRINSTD₁₇； D₄₀VDKNGSINAAD₅₁Mutation to D₄₀TSNNGTINAAD₅₁。

The preparation method of the fibrosome docking protein combined mutant 36864 comprises the following steps:

carrying out site-directed mutagenesis by taking a nucleotide sequence in the docking protein as shown in SEQ ID NO.3 to obtain a docking protein combination mutant 36864, designing a site-directed mutagenesis primer on the basis of the nucleotide sequence as shown in SEQ ID NO.1, carrying out PCR by taking a pET28a (+) vector carrying a cellosome docking protein gene as a template to construct a recombinant mutant plasmid, transforming the mutant plasmid into escherichia coli BL21(DE3), selecting a positive clone for fermentation, collecting thalli after the fermentation is finished, crushing the thalli, and purifying to obtain the cellosome docking protein mutant.

According to the preferable preparation method, the dockerin is mutated into a dockerin combination mutant 36864 by the dockerin with an amino acid sequence shown as SEQ ID No.4, and the amino acid sequence is shown as SEQ ID No. 2.

Preferably, in the above preparation method, after the fermentation is completed, the bacterial cells are collected by centrifugation, disrupted by sonication, and purified by affinity chromatography to obtain the mutant of the fibrosome dockerin.

According to the preferable preparation method, the PCR amplification of the dockerin combined mutant 36864 is carried out, the plasmid is divided into an AB section and a BA section, the AB section amplification primers are F1 and R2, the BA section amplification primers are F2 and R1, and the AB section and the BA section are respectively amplified by using the amplification primers; the nucleotide sequence of the PCR amplification primer is as follows:

F1：GTG gat GGT agc GGT cgt ATTAATAGCACCGAT SEQ ID NO.9；

R1：TTAAT acg ACC gct ACC atc CACGTCACCCAGCA SEQ ID NO.10；

F2：CGAT accagcaat AATGGC acc ATTAATGCCGCCGATGTTCT SEQ ID NO.11；

R2：ATTAAT ggt GCCATT attgctggt ATCGGCACGGGCTTTGGCA SEQ ID NO.12。

the lower case letters in the primer nucleotide sequence are mutation sites.

Further preferably, in the preparation method, the PCR reaction system:

plasmid DocA-pET28a vector template 1. mu.L, forward mutation primer F1/F22. mu.L, reverse mutation primer R2/R12. mu.L, 2 xMax Buffer 25. mu.L, dNTP 4. mu.L, DNA Polymerase 1. mu.L, ddH₂O 15μL。

Further preferably, in the preparation method, the PCR reaction conditions are as follows:

pre-denaturation at 95 ℃ for 3 min; 15sec at 95 ℃, 15sec at 60 ℃, 3min at 72 ℃ and 18 cycles; extension was supplemented at 72 ℃ for 7 min.

According to the invention, preferably, after the PCR reaction is completed, the original template in the PCR amplification product needs to be digested by Dnp I enzyme, and after the digestion reaction system is mixed uniformly, the original template is digested for 2 hours at 37 ℃.

Further preferably, the digestion system:

PCR amplification product 40. mu.L, Dnp I enzyme 1. mu.L.

Preferably, the digestion product is detected by DNA gel electrophoresis, and the AB section and the BA section are respectively recovered by using a glue recovery kit after detection.

According to the invention, preferably, the recovered AB section and BA section are recombined by utilizing Exnase II, the reaction product is the vector to be transformed, and the recombination reaction system is as follows:

ddH₂o10. mu.L, 5 XCE II Buffer 4. mu.L, product 2. mu.L recovered in AB, product 2. mu.L recovered in BA, and Exnase II 2. mu.L.

Further preferably, the transformation of the mutant vector comprises transforming the above-mentioned vector to be transformed into E.coli BL21(DE3) cells, and plating the transformant with a medium containing kanamycin (50. mu.g.mL)^-1) After overnight culture at 37 ℃ on the LB solid medium, single colonies are picked up, sequencing verification is carried out, and positive mutants are screened to obtain recombinant Escherichia coli 36864-BL 21.

More preferably, the mutant is expressed and purified by inoculating the correctly identified strain in liquid LB medium containing kanamycin (50. mu.g.mL-1), culturing overnight at 37 ℃, transferring to liquid LB medium, culturing at 37 ℃ until OD 600. apprxeq.1, adding to a final concentration of 1. mu.m.mL^-1The IPTG of (3), was induced at 26 ℃ for 8 hours at 200rpm, the induced cells were collected, then the cells were resuspended in 2 × PBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, the protein in the supernatant after centrifugation was purified using a nickel ion affinity column, and desalted by dialysis using PBS-EP + buffer, to obtain purified fibro-body-dockerin combinatorial mutant 36864.

The application of the fibrosome docking protein combination mutant 36864 in constructing a protein complex by interacting with the cadherin.

According to the invention, the use of the dockerin combination mutant 36864 for constructing protein complexes with mucin at low calcium ion concentrations is preferred.

Further preferably, the low calcium ion concentration is 10^-7M-10^-3M；

Further preferably, the low calcium ion concentration is 10^-7M-10^-4M；

Further preferably, the low calcium ion concentration is 10^-7M-10^-6M。

According to a preferred embodiment of the present invention, the dockerin combination mutant 36864 is used in constructing a protein complex by intracellular interaction with mucin.

The invention has the beneficial technical effects

1. The fibrosome dockerin combined mutant 36864 provided by the invention can reduce the calcium ion demand of the key element dockerin of the fibrosome, and realize the calcium ion environment in a wide range (the calcium ion concentration is 10)^-7M～2×10^-3M) effective binding to the fibronectin of the cellulosome and realizes the calcium ion concentration of 10^-7M～10^-6Under the condition of M, the protein can be effectively assembled with the fibronectin.

2. The mutant of the fibronectin realizes the effective assembly with the fibronectin in cells.

3. The invention provides a new method and a new way for assembling the intracellular multienzyme complex, and has wide application prospect.

Drawings

FIG. 1 is a schematic diagram of the separation of the plasmid into AB and BA segments in example 1;

FIG. 2 is a bar graph of intracellular interaction analysis of the mutant fibronectin and fibronectin corresponding to example 4;

FIG. 3 is a line graph showing the binding capacity of the mutant fibronectin of example 5 to mucin at different calcium ion concentrations.

Detailed Description

The invention is further illustrated with reference to specific examples, without limiting the scope of protection.

Sources of materials

The vectors DocA-pET28a and CohA-pET28a were extracted from Escherichia coli DH 5. alpha. or stored in the Escherichia coli DH 5. alpha. in the laboratory, which is an important laboratory in microbial engineering, Shandong, university of Qilu Industrial science, and those skilled in the art can construct the vectors according to the prior art or purchase them from the laboratory.

The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by the manufacturer, and are all common commercial products.

Example 1

Mutant primer design and mutant vector construction

Primer design of dockerin mutants was performed using dockerin DocA (vector DocA-pET28a, nucleotide sequence shown in SEQ ID NO. 13) ligated to pET28a (+) vector, respectively, as a template (see Table one). Wherein the DocA is derived from Clostridium thermocellum (Clostridia thermocellum) (GenBank:2CCL _ B), and the optimization of nucleotide sequence is carried out according to the codon preference of Escherichia coli, the DocA nucleotide sequence is shown as SEQ ID NO.3, and the DocA amino acid sequence is shown as SEQ ID NO. 4.

Watch-two-site mutation primer watch

Note: lower case letters are mutation sites.

The plasmid is divided into an AB section and a BA section by taking a site A to be mutated and a site B to be mutated as boundaries, and the AB section and the BA section are shown in a figure 1.

The AB fragment and BA fragment were amplified separately using Phanta enzyme.

The AB section amplification primers are F1 and R2, the BA section amplification primers are F2 and R1, the template plasmid is firstly digested by Dpn I after amplification, and the AB section and the BA section are respectively recovered after digestion. The PCR process, the Dpn I digestion process and the gel recovery process are as follows:

(1) and (3) PCR reaction, namely accurately adding the plasmid DocA-pET28a, the designed mutation primer, and the enzyme and buffer required by PCR according to the addition amount of each component in the second table, preparing PCR reaction solution of an experimental group (namely a double-site mutation group), and carrying out PCR reaction according to a third-table PCR reaction program.

Surface two-site mutation PCR reaction system

TABLE III PCR reaction conditions

(2) After the digestion of the amplification product, namely the PCR reaction is finished, in order to prevent the interference of a false positive transformant formed by the template plasmid DocA-pET28a after transformation, the plasmid DocA-pET28a is digested before a recombination cyclization experiment, the enzyme for digestion is Dpn I enzyme, the digestion reaction system is shown in the table four, and after the system is well configured and fully mixed, the system is digested for 2 hours at the temperature of 37 ℃.

TABLE IV digestion System

(3) And (3) recovering glue of the AB section and the BA section, namely performing DNA gel electrophoresis detection on the digestion product, and respectively recovering the AB section and the BA section by using a glue recovery kit after detection.

(4) And carrying out recombination reaction on the recovered AB section and BA section by utilizing Exnase II, adding each component in a sterile 200 mu L centrifugal tube in an ice bath according to the adding proportion of each component in the table five, gently blowing and uniformly mixing by using a pipette gun after the addition is finished, centrifuging for a short time by using a small centrifuge to avoid low recombination efficiency caused by residual components on the wall of the test tube, placing the centrifuged test tube in a metal water bath at 37 ℃ for accurate reaction for 30min, immediately placing the test tube in an ice box after the reaction is finished, and carrying out ice water bath for 5 min. The reaction product was either directly converted into competent BL21(DE3) or refrigerated in a refrigerator at-20 ℃.

TABLE V recombination reaction System

Mutant vector transformed host bacteria-taken out from-80 deg.C refrigeratorAppropriate amount of DH 5. alpha. competent cells were thawed by rapidly placing on ice, and after thawing, the procedure was performed according to the DH 5. alpha. competent transformation instructions, taking care that the time when the competent cells were just thawed was selected when the recombinant product was added, the time when the competent cells were just thawed was on ice except for heat shock, and the experimental work was gentle because the competent cells were easily inactivated, 400. mu.L of liquid LB medium (1% peptone, 1% yeast extract, 0.5% NaCl, and the balance water) was added to a clean bench after ice-bath, and then thawed in a shaker at 37 ℃ for about 1h, and after thawing, centrifugation at 4000rpm for 5 minutes was performed, and after removing 400. mu.L of supernatant, the balance 200. mu.L was directly applied to a medium containing kanamycin (50. mu.g.mL)^-1) After overnight culture on the solid medium (1% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, balance water), a single colony was picked up and cultured, and sent to sequencing company for sequencing and verifying the amino acid sequence D in DocA₆VNGDGTINSTD₁₇Mutation to D₆VDGSGRINSTD₁₇；D₄₀VDKNGSINAAD₅₁Mutation to D₄₀TSNNGTINAAD₅₁(ii) a Screening out positive clones to obtain recombinant Escherichia coli 36864-BL 21.

Example 2

Inducible expression and purification of muteins

The correctly verified strain was inoculated to a strain containing kanamycin (50. mu.g.mL)^-1) The cultured cells were cultured overnight at 37 ℃ in 50mL of liquid LB medium, and then transferred to a new 50mL of liquid LB medium at 37 ℃ in an amount of 2% by volume, and cultured to OD₆₀₀When the concentration is approximately equal to 1, the mixture is added until the final concentration is 1 mu m.mL^-1The IPTG was induced in a shaker at 26 ℃ and 200rpm for 8 hours, and the induced cells were collected. The induced cells were resuspended in 10mL 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, and the protein in the supernatant after centrifugation was purified by a nickel ion affinity column and desalted by dialysis using PBS-EP + buffer (from GE). Purified cellulosome dockerin combinatorial mutant 36864 was obtained.

Example 3

Construction and induced expression of expression vector of fibronectin

Transformation of the CohA-pET28a vector was carried out in the same manner as in example 1 using pET28a (+) vector to which the cellulosome fibronectin CohA was ligated, and similarly transformation of DocA-pET28a vector was prepared to obtain recombinant E.coli DocA-BL 21 and CohA-BL21, and inducible expression and purification of genes were carried out in the same manner as in example 2. Purified fibrosome mucin CohA, fibrosome dockerin DocA was obtained. Wherein the cellulosome mucin CohA is derived from a Clostridium thermocellum (Clostridium thermocellum) scaf gene (GenBank: MH049738.1), the nucleotide sequence of the CohA is shown as SEQ ID NO.5, and the amino acid sequence of the CohA is shown as SEQ ID NO. 6.

Example 4

Intracellular interaction analysis of fibronectin and fibronectin

The intracellular fibrosome dockerin-mucin interaction was characterized using bimolecular fluorescence complementation (BIFC). The BIFC uses fluorescent protein eYFP, the nucleotide sequence of the eYFP is shown as SEQ ID NO.7, and the amino acid sequence of the eYFP is shown as SEQ ID NO. 8. The double expression plasmid pETDuet-1 of Escherichia coli is taken as a vector, and the fluorescent protein eYFP is split into two sections of polypeptides from amino acid position 155, namely eYN (1-155) and eYC (156-238).

The two fluorescent complementary fragments are respectively connected with the dockerin and the cohesin through flexible connecting peptide SGGGSGGGSGGS, and are fused with the proteins according to a certain sequence, and the two fluorescent complementary fragments are taken as two independent coding proteins to be simultaneously expressed on pETDuet-1. The plasmid pETDuet-1-eYN (1-155) -CohA-DocA-eYC (156-238) was obtained and transformed by the transformation method described in example 1. Interaction of the dockerin with the fibronectin brings the eYN (1-155) and eYC (156-238) fragments fused to it in close spatial proximity to each other, which brings the two non-fluorescing fragments back together to reform the fluorescent protein eYFP, which is excited under 488nm light, said fusion sequence being synthesized by Biotechnology (Shanghai) GmbH.

Constructing recombinant plasmids corresponding to the dockerin combination mutant 36864 and the mucin CohA pair in the same way; transformation of the pETDuet-1 vector was performed as in example 1.

Finally, coexpression recombination BL21 was obtained, pETDuet-1-eYN (1-155) -CohA-DocA-eYC (156-238) and BL21, pETDuet-1-eYN (1-155) -CohA-36864-eYC (156-238), and the blank control was E.coli BL21, and the gene was expressed by induction in the same manner as in example 2. The Abs value (i.e., fluorescence intensity value) was quantitatively analyzed by fluorescent protein using a microplate reader, and the results are shown in fig. 2.

FIG. 2 shows that the fluorescence excitation values of DocA fused with BIFC protein and control bacteria are close, indicating that the non-mutated DocA and CohA can not effectively interact in the low calcium ion environment in the cell. The fluorescence excitation value of the dockerin combined mutant 36864 fused with the BIFC protein is obviously higher than that of DocA and a control bacterium, which shows that the requirement of the dockerin of key elements of the cellosome on calcium ions is reduced by mutating the sequence with the calcium ion binding function in the cellosome dockerin, and the dockerin is promoted to be in a low calcium ion concentration (the calcium ion concentration is less than or equal to 10) in cells^-6M) and the fibronectin to assemble, and lays a foundation for the intracellular assembly of the fibrosomes.

Example 5

Analysis of binding Capacity of Fibrosomal docking protein and mucin at different calcium ion concentrations

Analyzing the binding capacity between the mutant of the cellosome docking protein and the fibronectin with different calcium ion concentrations by using a biomacromolecule interaction instrument, selecting a proper CM5 chip as an anchoring chip, calculating the approximately required concentration of the protein CohA according to the formula, performing gradient dilution on acetic acid-sodium acetate buffer solutions with different pH values according to the approximately protein concentration to serve as the protein to be anchored, determining the optimal anchoring concentration and pH value according to the anchoring condition, and then anchoring the CohA according to the fibronectin in Biacore micromolecule application operation manual. The anchored chip was loaded into a molecular interaction apparatus in 1 XPBS-HP + solution (available from GE), and the dockerin and mutant were diluted to nearly the same anchoring concentration as the protein CohA and combined with different concentrations of CaCl₂Standing at 4 deg.CAfter 30min, the binding was determined on the machine and judged according to the AbsResp value (i.e., the binding interaction intensity value), and the results are shown in fig. 3 (table six). This result indicates that dockerin combinatorial mutant 36864 has calcium concentration > 10 relative to unmutated dockerin DocA^-4The binding capacity of M and the mucoprotein CohA is obviously enhanced, and the result simultaneously discovers that the dockerin combination mutant 36864 has the calcium ion concentration less than or equal to 10^-4M still has strong binding capacity with the mucoprotein CohA, which indicates that the dockerin combined mutant 36864 can be used in a wide range of calcium ion environments (calcium ion concentration is 10)^-7M～2×10^-3) Play a role in the process. Furthermore, although the unmutated dockerin DocA was found to have a calcium ion concentration of 10 or less^-6M has a certain binding signal with the mucin CohA, and the AbsResP value is about 200, but the result of the binding example 4 shows that the binding can not exist continuously and stably, and the binding is invalid, and the mutant protein and the mucin AbsResP value exceeds 400, which shows that the two proteins can be stably combined together. Research finds that the dockerin combination mutant 36864 has calcium ion concentration over 10^-3The activity of M is reduced to a certain extent, which shows that the structure of the mutant site region can be changed when the calcium ion concentration is too high, and the performance of the docking capability of the mutant protein is influenced.

Analysis of binding ability of the mutants to mucin at different calcium ion concentrations

The fibrosome dockerin combined mutant 36864 provided by the invention can reduce the calcium ion demand of the key element dockerin of the fibrosome, and realize the calcium ion environment in a wide range (the calcium ion concentration is 10)^-7M～2×10^-3M) effective binding to the fibronectin of the cellulosome and realizes the calcium ion concentration of 10^-7M～10^-6Can be effectively assembled with the fibronectin under the condition of M; the effective assembly of the adhesion protein with the cellulosome in the cell is realized; the invention provides a novel method and a novel way for assembling intracellular multi-enzyme complexes, and has wide application rangeThe application prospect of (1).

The fibrosome docking protein in the prior art contains calcium ion binding sites, the formation of the active structure of the fibrosome docking protein needs the support of calcium ions, the active structure interacts with the calcium ions and needs high calcium ion concentration, and then the fibrosome docking protein forms a specific conformation and interacts with the fibrosome fibronectin, and then the assembly of the whole fibrosome is completed. The dockerin combined mutant 36864 obtained by multi-site fixed-point mutation can form a specific conformation under the condition of low calcium ion concentration, and interacts with the fibronectin of the cellulosome to complete the assembly of the whole cellulosome; and interaction assembly can be realized in cells; which would not have been expected by the person skilled in the art.

SEQUENCE LISTING

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aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaa 717

<210> 8

<211> 238

<212> PRT

<213> Artificial sequence

<400> 8

Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

20 25 30

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe

50 55 60

Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Leu

65 70 75 80

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

85 90 95

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence

<400> 9

gtggatggta gcggtcgtat taatagcacc gat 33

<210> 10

<211> 34

<212> DNA

<213> Artificial sequence

<400> 10

ttaatacgac cgctaccatc cacgtcaccc agca 34

<210> 11

<211> 42

<212> DNA

<213> Artificial sequence

<400> 11

cgataccagc aataatggca ccattaatgc cgccgatgtt ct 42

<210> 12

<211> 43

<212> DNA

<213> Artificial sequence

<400> 12

attaatggtg ccattattgc tggtatcggc acgggctttg gca 43

<210> 13

<211> 5528

<212> DNA

<213> Artificial sequence

<400> 13

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atggtgctgc tgggtgacgt gaatggtgac 5100

ggtaccatta atagcaccga tctgaccatg ctgaaacgtt ctgttctgcg tgccattacc 5160

ctgaccgatg atgccaaagc ccgtgccgat gtggataaaa atggcagcat taatgccgcc 5220

gatgttctgc tgctgtctcg ctatctgctg cgtgttattg ataaaggagg aggcggctcg 5280

ggaggaggcg gctcgggagg aggcggctcg catcatcatc atcatcatta agaattcgag 5340

ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 5400

tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 5460

ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 5520

atccggat 5528

Claims

1. A cellosome docking protein combination mutant 36864 suitable for low calcium ion concentration is characterized in that, a cellosome docking protein DocA, the nucleotide sequence is as shown in SEQ ID NO.3, and site-directed mutagenesis is carried out , to obtain a docking protein combination mutant 36864 suitable for low calcium ion concentration, and the nucleotide sequence is shown in SEQ ID NO.1.

2. A cellulosome docking protein combination mutant 36864 suitable for low calcium ion concentration, characterized in that a cellosome docking protein DocA, the amino acid sequence of which is shown in SEQ ID NO.4, is subjected to site-directed mutation to obtain A docking protein combination mutant 36864 suitable for low calcium ion concentration, the amino acid sequence is shown in SEQ ID NO.2.

3. the preparation method of mutant 36864 as described in any one of claim 1-2, is characterized in that, comprises the steps:

Based on the nucleotide sequence in the above docking protein as shown in SEQ ID NO.3, site-directed mutagenesis was performed to obtain the docking protein combination mutant 36864, and the nucleotide sequence was shown in SEQ ID NO.1 as the basis to design site-directed mutagenesis primers , using the pET28a(+) vector carrying the cellulosome docking protein gene as a template, carry out PCR, construct a recombinant mutant plasmid, and transform the mutant plasmid into Escherichia coli BL21 (DE3), select positive clones for fermentation, and collect after fermentation The cells were broken and purified to obtain cellulosome docking protein mutants.

4. The preparation method of mutant 36864 as claimed in claim 3, characterized in that, in the preparation method, a docking protein whose amino acid sequence is shown in SEQ ID NO. 4 is mutated into a docking protein combination mutant 36864, whose amino acid The sequence is shown in SEQ ID NO.2.

5. The preparation method of mutant 36864 as claimed in claim 3, characterized in that, after the fermentation is completed, the cells are collected by centrifugation, the cells are subjected to ultrasonic breakage, and purified by affinity chromatography to obtain the cellosome docking protein mutant.

6. the preparation method of mutant 36864 as claimed in claim 3, is characterized in that, the PCR amplification of docking protein combination mutant 36864, plasmid is divided into AB section and BA section, and AB section amplification primer is SEQ ID NO. 9 and SEQ ID NO. 12, the BA segment amplification primers are SEQ ID NO. 11 and SEQ ID NO. 10, and the AB segment and the BA segment are amplified respectively by using the amplification primers.

7. the preparation method of mutant 36864 as claimed in claim 6, is characterized in that, PCR reaction system:

Plasmid DocA-pET28a vector template 1 μL, forward mutation primer F1/F2 2 μL, reverse mutation primer R2/R1 2 μL, 2× Max Buffer 25 μL, dNTP 4 μL, DNA Polymerase 1 μL, ddH ₂ O 15 μL.

8. the preparation method of mutant 36864 as claimed in claim 6, is characterized in that, in described preparation method, PCR reaction condition:

Pre-denaturation at 95 °C for 3 min; 18 cycles of 95 °C for 15 sec, 60 °C for 15 sec, 72 °C for 3 min; supplementary extension at 72 °C for 7 min.

9. the preparation method of mutant 36864 as claimed in claim 6 is characterized in that, after PCR reaction is completed, need to use Dnp I enzyme to digest the original template in the PCR amplification product, after the digestion reaction system is mixed, the It was digested at 37°C for 2h.

10. The preparation method of mutant 36864 as claimed in claim 9, wherein,

The digestive system:

PCR amplification product 40 μL, Dnp I enzyme 1 μL.

11 . The method for preparing mutant 36864 according to claim 9 , wherein the digestion product is subjected to DNA gel electrophoresis detection, and after detection, the AB segment and the BA segment are respectively recovered by a gel recovery kit. 12 .

12. the preparation method of mutant 36864 as claimed in claim 11, is characterized in that, utilizes Exnase II to carry out the reorganization of AB section after recovery and BA section, and reaction product is the carrier to be transformed, and the recombination reaction system is as follows:

10 μL of ddH ₂ O, 4 μL of 5× CE II Buffer, 2 μL of recovered product from AB segment, 2 μL of recovered product from BA segment, and 2 μL of Exnase II.

13. The preparation method of mutant 36864 as claimed in claim 12, characterized in that, for the transformation of the mutant vector, the above-mentioned vector to be transformed is transformed into Escherichia coli BL21 (DE3) cells, and the transformant is coated on a cell containing kanamyces After culturing overnight at 37°C on LB solid medium containing 50 μg·mL ^-1 of glutathione, single colonies were picked and verified by sequencing, and positive mutants were screened to obtain recombinant Escherichia coli 36864-BL21.

14. The preparation method of mutant 36864 as claimed in claim 13, characterized in that, for the expression and purification of the mutant, the above-mentioned correct bacterial strain is inoculated into the liquid LB medium containing kanamycin 50 μg·mL ⁻¹ medium, cultured at 37°C overnight, then transferred to liquid LB medium at 37°C and cultured until OD600≈1, added IPTG with a final concentration of 1 μm·mL ^-1 , induced at 26°C and 200rpm for 8 hours, and collected After induction, the cells were resuspended in 2ⅹPBS buffer, disrupted by sonication, centrifuged at 10,000 rpm for 10 min, and the protein in the supernatant after centrifugation was purified by nickel ion affinity column and dialyzed against PBS-EP+ buffer Desalting yielded purified cellulosome docking protein combinatorial mutant 36864.

15. the application of the mutant 36864 described in any one of claim 1-2 in interacting with cohesin CohA in low calcium ion concentration to construct the protein complex, and described low calcium ion concentration is 10 ^-7 M - 10 ^{- 3M} .

16. The application according to claim 15, wherein the low calcium ion concentration is 10 ^-7 M - 10 ^-4 M.

17. The application according to claim 16, wherein the low calcium ion concentration is 10 ^-7 M - 10 ^-6 M.

18. The application of claim 15, wherein the docking protein combination mutant 36864 interacts with cohesin CohA to construct a protein complex in cells.