CN111848758A - Cellulosome docking protein mutant suitable for low calcium ion concentration and application - Google Patents

Cellulosome docking protein mutant suitable for low calcium ion concentration and application Download PDF

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CN111848758A
CN111848758A CN202010730636.7A CN202010730636A CN111848758A CN 111848758 A CN111848758 A CN 111848758A CN 202010730636 A CN202010730636 A CN 202010730636A CN 111848758 A CN111848758 A CN 111848758A
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汪俊卿
李楠
薛乐
范翰
王瑞明
李丕武
姜彦君
王子睿
王大涛
包涵
刘炜杰
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Qilu University of Technology
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Abstract

The invention discloses a small-fiber docking protein mutant suitable for low calcium ion concentration and application thereof, wherein the small-fiber docking protein mutant suitable for low calcium ion concentration is characterized in that D mutation of a D1 site in a D1D/N3D/N5D/N9D 12 amino acid sequence in docking protein is R, namely the mutation is an R1D/N3D/N5D/N9D 12 amino acid sequence; the butt jointProtein mutant 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

Cellulosome docking protein mutant suitable for low calcium ion concentration and application
The technical field is as follows: the invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a cellosome docking protein mutant suitable for low calcium ion concentration and application thereof.
Background
The cellulosome is a natural multienzyme self-assembly system which can organize and coordinate a plurality of enzyme components to efficiently catalyze and degrade lignocellulose in a synergetic way, and the cellulosome mainly comprises two parts: dockerin (Doc), which contains enzymes or other accessory proteins, and fibronectin (cohesin, Coh), which contains structural proteins, may also be referred to in a broad sense as scaffold proteins. The cellosome is used as a natural and efficient multienzyme self-assembly system, the extracellular surface display is successfully realized in various microorganisms such as bacteria, yeast and the like at present, proteins or enzymes with different functions are fused and connected on Doc and Coh by utilizing the technology of fusion enzyme, a multienzyme complex with various proteins or enzymes close to each other is constructed by utilizing the principle that Doc and Coh attract and assemble with each other, the transmission distance of substances between the enzymes is shortened, the reaction efficiency of multienzyme series reaction is further improved, and the application is realized in various fields such as biomass degradation, ethanol and 2, 3-butanediol synthesis, biosensors and the like [ bulb, journal Biological Chemistry, 2016 and 29; liang, Applied & Environmental Microbiology,2014,80(21): 6677-6684.
The research of the prior art finds that a sequence D rich in hydrophilic amino acid exists in a cellulosome element Doc1D/N3D/N5D/N9D12And 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 mutant of cellosome dockerin 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 mutant of fibrosome docking protein suitable for low calcium ion concentration is D in docking protein1D/N3D/N5D/N9D12D in amino acid sequence1The mutation of D to R of the site, i.e. to R1D/N3D/N5D/N9D12An amino acid sequence.
D is aspartic acid, N is asparagine, and R is arginine; the superscript number indicates that the site is at the position of the sequence, and the cellulosome dockerin pseudomutation amino acid site D1Is characterized in that: d1The last 3, 5 and 9 amino acids are D or N, D1The latter 12 th amino acid is D.
A nucleotide sequence of a fibrosome docking protein is shown as SEQ ID NO.1, an amino acid sequence is shown as SEQ ID NO.3, fixed point mutation is carried out to obtain a docking protein mutant, the nucleotide sequence is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 7.
The DVDKNGSINSTD mutation in the above amino acid sequence was RVDKNGSINSTD.
A nucleotide sequence of a fibrosome docking protein is shown as SEQ ID NO.2, an amino acid sequence is shown as SEQ ID NO.4, fixed point mutation is carried out to obtain a docking protein mutant, the nucleotide sequence is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO. 8.
The DLNGDGNINSSD mutation in the above amino acid sequence was RLNGDGNINSSD.
The preparation method of the mutant of the fibrosome docking protein comprises the following steps:
as D in the above dockerin1D/N3D/N5D/N9D12Amino acid sequence based according to D1D mutation of the site is R, a site-directed mutation primer is designed, a pET28a (+) vector carrying a cellosome docking protein gene is used as a template, PCR is carried out, recombinant mutant plasmids are constructed, the mutant plasmids are converted into escherichia coli BL21(DE3), positive clones are selected for fermentation, thalli are collected after fermentation is finished, and the thalli are crushed and purified to obtain the cellosome docking protein mutant.
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 invention, the preparation method comprises the following steps:
designing primers of a docking protein mutant by taking docking protein DocA (vector is DocA-pET28a) or DocB (vector is DocB-pET28a) which are respectively connected between the NcoI vector and the EcoRI restriction enzyme sites of the pET28a (+) vector as a template, carrying out PCR (polymerase chain reaction), and constructing corresponding mutant plasmids; wherein the dockerin DocA is derived from Clostridium thermocellum (Clostridium thermocellum) xynY gene (GenBank: X83269.1), the DocB is derived from Clostridium thermocellum (Clostridium thermocellum) celQ gene (GenBank: AB047845.2), the nucleotide sequence of the DocA is shown as SEQ ID NO.1, and the nucleotide sequence of the corresponding mutant D-RA is shown as SEQ ID NO. 5; or the nucleotide sequence of DocB is shown as SEQ ID NO. 2; the nucleotide sequence of the corresponding mutant D-RB is shown as SEQ ID NO. 6; and transforming the mutant plasmid into escherichia coli BL21(DE3), selecting positive clone for fermentation, collecting thalli after fermentation is finished, crushing the thalli, and purifying to obtain the mutant of the fibrosome docking protein.
Further preferably, in the preparation method, the dockerin DocA amino acid sequence is shown as SEQ ID NO.3, the dockerin DocA amino acid sequence is mutated into a mutant D-RA, and the dockerin DocA amino acid sequence is shown as SEQ ID NO. 7; or the dockerin DocB has an amino acid sequence shown in SEQ ID NO.4, is mutated into a mutant D-RB, and has an amino acid sequence shown in SEQ ID NO. 8;
the DVDKNGSINSTD mutation in the dockerin DocA amino acid sequence was RVDKNGSINSTD.
The DLNGDGNINSSD mutation in the dockerin DocB amino acid sequence was RLNGDGNINSSD.
Further preferably, in the preparation method, the nucleotide sequence of the PCR amplification primer of the dockerin DocA mutant is as follows:
D-RA-F1：CAAAGGCTAGAGCAagaGTTGACAAGAATGGATCG SEQ ID NO.13
D-RA-R1：CCATTCTTGTCAACtctTGCTCTAGCCTTTGCATC SEQ ID NO.14
the nucleotide sequence of the PCR amplification primer of the dockerin DocB mutant is as follows:
D-RB-F1：AATTGAAAGCTGCTagaCTTAACGGAGATGGCAAT SEQ ID NO.15
D-RB-R1：CCATCTCCGTTAAGtctAGCAGCTTTCAATTTAAC SEQ ID NO.16
the lower case letters in the primer nucleotide sequence are mutation sites.
Further preferably, in the preparation method, the PCR reaction system:
1 μ L of plasmid vector template, 2 μ L of forward mutation primer F, 2 μ L of reverse mutation primer R, 10 μ L of 5 XFastmutation buffer, 1 μ L of Fastmutation DNA Polymerase, ddH2O34μL。
Further preferably, in the preparation method, the PCR reaction conditions are as follows:
pre-denaturation at 95 ℃ for 2 min; 94 ℃ for 20sec, 55 ℃ for 10sec, 68 ℃ for 3min, 18 cycles; extension was supplemented at 68 ℃ for 5 min.
According to the optimization of the invention, after the PCR reaction is finished, the original template in the PCR amplification product needs to be digested by DnpI enzyme, and after the digestion reaction system is mixed uniformly, the original template is digested for 1 hour at 37 ℃ to obtain the vector to be transformed.
Further preferably, the digestion system:
PCR amplification product 49. mu.L, DnpI enzyme 1. 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 LB solid medium, a single colony was picked and culturedSequencing verification is carried out, and positive mutants are screened to obtain recombinant escherichia coli D-RA-BL21 and D-RB-BL21 containing the genes of the fibrosome docking protein mutant D-RA and D-RB respectively.
More preferably, the mutant is expressed and purified by inoculating the correctly verified strains separately into a culture containing kanamycin (50. mu.g.mL)-1) The cultured cells were cultured overnight at 37 ℃ in the liquid LB medium, and then transferred to the liquid LB medium and cultured to OD at 37 ℃600When the concentration is approximately equal to 1, the mixture is added until the final concentration is 1 mu m.mL-1The IPTG was induced at 26 ℃ for 8 hours at 200rpm, the induced cells were collected, resuspended in 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 with PBS-EP + buffer to obtain purified mutant cellulosome-dockerin D-RA and D-RB, respectively.
The application of the mutant of the cellulosome docking protein in constructing a protein complex by interacting with the mucin.
According to a preferred embodiment of the invention, the mutant dockerin interacts with mucin at low calcium ion concentrations to construct a protein complex.
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 invention, the dockerin mutant interacts with fibronectin in the cell to construct a protein complex.
The fibrosome docking protein can be specifically combined with the fibrosome cadherin.
The cellulosome fibronectin does not contain a calcium ion binding site, and the formation of an active structure of the fibronectin does not need the support of calcium ions.
The fibrosome docking protein contains calcium ion bindingThe formation of the site, its active structure, requires the support of calcium ions. I.e. the calcium ion binding function sequence D of the presence of the fibrosome docking protein1D/N3D/N5D/N9D12Can rely on D1、D/N3、 D/N5And D12The hydrophilic amino acids at the four sites interact with calcium ions simultaneously, so that the fibrosome dockerin forms a specific conformation and interacts with the fibrosome cohesin, and the assembly of the whole fibrosome is completed.
Site D to which the present invention relates1Aspartic acid, which has negative charge under physiological pH value and can be combined with calcium ions, and simultaneously, the interaction occurs, so that the fibrin forms a specific conformation; the mutation is arginine, which has positive charge under physiological pH value, is repelled from calcium ions and can not combine with calcium ions; the invention relates to a mutation site which can make the cellulosome dockerin form a specific conformation under the condition of not combining calcium ions and interact with the cellulosome adhesive protein to complete the assembly of the whole cellulosome; and can be assembled in intracellular interactions; which would not have been expected by the person skilled in the art.
The invention has the advantages of
1. The mutant of the fibrosome docking protein reduces the requirement of the key element of the fibrosome docking protein on calcium ions, and realizes 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 histogram of intracellular interaction analysis of the mutant fibronectin and fibronectin corresponding to example 4
FIG. 2 is a line graph showing the binding capacity of the mutant fibronectin and fibronectin at different calcium ion concentrations in example 5
Detailed Description
The invention is further illustrated with reference to specific examples, without limiting the scope of protection.
Sources of materials
Vectors DocA-pET28a, DocB-pET28a and CohA-pET28a were extracted or stored from E.coli DH 5. alpha. and were provided by the key laboratory of microbial engineering, Shandong, university of Qilu Industrial science, and those skilled in the art either constructed according to the prior art or purchased from the key 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
Designing primers of a docking protein mutant (shown in table I) by using docking protein DocA (the vector is DocA-pET28a, the nucleotide sequence of which is shown in SEQ ID NO. 17) or DocB (the vector is DocB-pET28a, the nucleotide sequence of which is shown in SEQ ID NO. 18) which is respectively connected between restriction sites of a pET28a (+) vector NcoI and EcoRI as a template, carrying out PCR (polymerase chain reaction) to construct corresponding mutant plasmids; wherein the dockerin DocA is derived from Clostridium thermocellum (Clostridium thermocellum) xynY gene (GenBank: X83269.1), the DocB is derived from Clostridium thermocellum (Clostridium thermocellum) celQ gene (GenBank: AB047845.2), the nucleotide sequence of DocA is shown as SEQ ID NO.1, the nucleotide sequence of DocB is shown as SEQ ID NO.2, the amino acid sequence of DocA is shown as SEQ ID NO.3, and the amino acid sequence of DocB is shown as SEQ ID NO. 4.
The calcium ion binding site sequence contained in DocA was "DVDKNGSINSTD", and the calcium ion binding site sequence contained in DocB was "DLNGDGNINSSD".
TABLE-mutant primer Table
Figure BDA0002603106800000061
Note: lower case letters are mutation sites.
The nucleotide sequence of the dockerin mutant D-RA is shown as SEQ ID NO.5, the nucleotide sequence of the dockerin mutant D-RB is shown as SEQ ID NO.6, the amino acid sequence of the dockerin mutant D-RA is shown as SEQ ID NO.7, and the amino acid sequence of the dockerin mutant D-RB is shown as SEQ ID NO. 8. And accurately adding plasmids DocA-pET28a or DocB-pET28a, the designed mutation primers, PCR enzyme and buffer according to the addition amount of each component in the second table to prepare a PCR reaction solution, and carrying out PCR amplification according to the PCR reaction conditions in the third table.
PCR reaction system for epidiaschisis
Figure BDA0002603106800000062
PCR reaction conditions for the epitrimutation
Figure BDA0002603106800000063
Figure BDA0002603106800000071
After the PCR reaction is completed, original plasmid templates of Escherichia coli DH5 alpha methylated DocA-pET28a and DocB-pET28a in the PCR product are digested by Dnp I enzyme respectively, the digestion reaction system is shown in the fourth table, and after the system is prepared and fully mixed, the mixture is digested for 1h at 37 ℃.
TABLE IV digestion System
Figure BDA0002603106800000072
Transformation of the mutant vector
Taking out Escherichia coli BL21(DE3) at-80 deg.C, rapidly dissolving in ice at 4 deg.C, sucking appropriate amount of the above enzyme solution with micropipette, and injecting into melted large intestineIn bacillus BL21(DE3) competence, the tube wall is uniformly mixed, then heat shock is accurately carried out for 90sec at 42 ℃, ice bath is carried out for 2min, 400 microliter of liquid LB culture medium (1 percent of peptone, 1 percent of yeast extract powder, 0.5 percent of NaCl and the balance of water) is added into an ultra-clean workbench after ice bath, recovery is carried out for about 1h in a shaking table at 37 ℃, centrifugation is carried out for 5min at 4000rpm after recovery is finished, 400 microliter of supernatant is removed, and the balance of 200 microliter is directly coated to contain kanamycin (50 microgram mL. mL)-1) After overnight culture on the solid culture medium (1% peptone, 1% yeast extract, 0.5% NaCl, 2% agar and the balance water), selecting a single colony for culture, sending the single colony to a sequencing company for sequencing verification to verify whether the sequence DVDKNGSINSTD in the DocA and the sequence DLNGDGNINSSD in the DocB are mutated into RVDKNGSINSTD and RLNGDGNINSSD respectively, and screening out positive clones. Finally, recombinant escherichia coli D-RA-BL21 and D-RB-BL21 containing the genes of the fibrosome docking protein mutant D-RA and D-RB respectively are obtained.
Example 2
Inducible expression and purification of muteins
The strains verified to be correct in example 1 were inoculated to a medium containing kanamycin (50. mu.g. mL)-1) The cultured cells are cultured overnight at 37 ℃ in 50mL of liquid LB medium, then respectively transferred to a new 50mL of liquid LB medium for culture at 37 ℃ according to the volume percentage of 2 percent, and cultured until OD is reached600When the concentration is approximately equal to 1, the mixture is added until the final concentration is 1 mu m.mL-1The IPTG was placed in a shaker at 26 ℃ and 200rpm for 8 hours, and the induced cells were collected. The induced thallus was resuspended in 10mL of 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, and the protein in the supernatant after centrifugation was purified by nickel ion affinity column and desalted by dialysis with PBS-EP + buffer (from GE Co.); purified mutant fibrinosomal dockerin D-RA and D-RB were obtained, respectively.
Example 3
Construction and induced expression of expression vector of fibronectin
Plasmids DocA-pET28a, DocB-pET28a and CohA-pET28a, in which the cellosome mucin CohA was derived from Clostridium thermocellum (Clostridium thermocellum) craft gene (GenBank: MH049738.1), were extracted as the subjects of investigation, respectively, and the CohA gene was ligated between pET28a (+) vector NcoI and EcoRI cleavage sites. The nucleotide sequence of CohA is shown in SEQ ID NO.9, and the amino acid sequence of CohA is shown in SEQ ID NO. 10.
Taking Escherichia coli BL21(DE3) competence at-80 ℃ and quickly dissolving the Escherichia coli BL21(DE3) competence on ice at 4 ℃, respectively sucking a proper amount of DocA-pET28a, DocB-pET28a and CohA-pET28a plasmids by a micropipette, injecting the plasmids into the molten Escherichia coli BL21(DE3) competence, uniformly mixing the walls of a flick tube, accurately thermally shocking the flick tube for 90sec at 42 ℃, carrying out ice bath for 2min, adding 400 mu L of liquid LB culture medium (1% peptone, 1% yeast extract powder, 0.5% NaCl and the balance of water) into an ultraclean workbench after the ice bath, recovering the mixture in a shaker at 37 ℃ for about 1h, centrifuging the mixture for 5min at 4000rpm after the recovery is finished, removing 400 mu L of supernatant, and directly coating the balance of 200 mu L of the mixture to a mixture 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), single colony culture was picked and sent to sequencing company to verify whether mutation was successful. Recombinant E.coli DocA-BL21, DocB-BL21 and CohA-BL21 were obtained.
The correctly confirmed strains were inoculated to a medium containing kanamycin (50. mu.g.mL)-1) The 50mL of liquid LB medium is cultured overnight at 37 ℃, then is respectively transferred to a new 50mL of liquid LB medium for culture at 37 ℃ according to the volume percentage of 2 percent, and is added with 1 μm/mL when the culture is carried out until OD600 is approximately equal to 1-1The IPTG was placed in a shaker at 26 ℃ and 200rpm for 8 hours, and the induced cells were collected. The induced cells were resuspended in 10mL of 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, and the proteins in the supernatant after centrifugation were purified by nickel ion affinity column, dialyzed against PBS-EP + buffer (available from GE Co.) to remove salts. Purified cellulosome dockerin, DocB and cohesin CohA were obtained.
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.11, and the amino acid sequence of the eYFP is shown as SEQ ID NO. 12. 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 were fused to the C-terminal of dockerin (DocA-eYC (156-238)) and the N-terminal of cohesin (eYN (1-155) -CohA) via flexible linker SGGGSGGGSGGS, respectively, and expressed on pETDuet-1 as two independently encoded proteins at the same time to construct pETDuet-1-eYN (1-155) -CohA-DocA-eYC (156-238) plasmid, which was transformed according to 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 thereto in close spatial proximity to each other, which brings the two non-fluorescent fragments back together to reform the fluorescent protein eYFP, which is then excited under 488nm light.
Constructing recombinant plasmids corresponding to dockerin DocB, mutant D-RA and mutant D-RB and cohesin CohA in the same way;
finally, coexpression recombinant bacteria BL21: pETDuet-1-eYN (1-155) -CohA-DocA-eYC (156-. The Abs value (i.e., fluorescence intensity value) was quantitatively analyzed for fluorescent proteins using a microplate reader, and the expression results of each protein under the above conditions are shown in fig. 1.
As shown in FIG. 1, the fluorescence excitation values of DocA and DocB fused with BIFC proteins are close to those of control bacteria, indicating that neither of the non-mutated DocA and DocB and CohA can effectively interact in the intracellular low calcium environment. The fluorescence excitation values of D-RA and D-RB fused with BIFC protein are obviously higher than those of DocA and DocB fused with BIFC protein and control bacteria, which shows that the requirement of the cellulose body key element docking protein on calcium ions is reduced by mutating the sequence with calcium ion binding function in the cellulose body docking protein, and the docking protein is promoted to be in 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 further application of the assembly of the cellulosome intracellular.
Example 5
Analysis of binding Capacity of Fibrosomal docking protein and mucin at different calcium ion concentrations
Figure BDA0002603106800000091
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 1PBS-HP + solution (from GE), the dockerin and mutants were diluted to the same concentration as the protein CohA, and different concentrations of CaCl were combined2After standing at 4 ℃ for 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. 2 (table 1). The results show that the mutant protein D-RA and mutant protein D-RB are at calcium concentrations > 10 relative to the unmutated dockerin DocA and DocB-4The binding capacity of the protein M to the mucoprotein CohA is obviously enhanced, and the results show that the mutant protein D-RA and the mutant protein D-RB have the calcium ion concentration of less than or equal to 10-4M still has a certain binding capacity with the mucoprotein CohA, which indicates that the mutant protein D-RA and the mutant protein D-RB can be in a wide range of calcium ion environments (the calcium ion concentration is 10)-7M～2×10-3M) is used. Furthermore, although the unmutated dockerin DocA and DocB proteins were found to have a calcium ion concentration of 10 or less-6M has a certain binding signal with the mucoprotein CohA, the value of the AbsResP is about 300, but the result of the binding example 4 shows that the binding can not exist continuously and stably at the moment and is invalid,whereas, the values of the mutant protein and the cohesin AbsResP exceed 400, which indicates that the two proteins can be stably combined together. The research finds that the concentration of calcium ions of the mutant protein D-RA and the mutant protein D-RB exceeds 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.
TABLE 1 analysis of the binding Capacity of the mutant fibronectin and fibronectin at different calcium ion concentrations
Figure 1
The mutant of the fibrosome docking protein reduces the requirement of the key element of the fibrosome docking protein on calcium ions, and realizes 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 new method and a new way for assembling the intracellular multienzyme complex, and has wide application prospect.
Site D to which the present invention relates1Aspartic acid, which has negative charge under physiological pH value and can be combined with calcium ions, and simultaneously, the interaction occurs, so that the fibrin forms a specific conformation; the mutation is arginine, which has positive charge under physiological pH value, is repelled from calcium ions and can not combine with calcium ions; the invention relates to a mutation site which can make the cellulosome dockerin form a specific conformation under the condition of not combining calcium ions and interact with the cellulosome adhesive protein to complete the assembly of the whole cellulosome; and can be assembled in intracellular interactions; which would not have been expected by the person skilled in the art.
SEQUENCE LISTING
<110> university of Qilu Industrial science
<120> a mutant of cellosome docking protein suitable for low calcium ion concentration and application
<160>18
<170>PatentIn version 3.5
<210>1
<211>261
<212>DNA
<213> Artificial sequence
<400>1
atggtactgc ttggcgatgt aaacggtgat ggaaccatta actcaactga cttgacaatg 60
ttaaagagaa gcgtgttgag ggcaatcacc cttaccgacg atgcaaaggc tagagcagac 120
gttgacaaga atggatcgat aaacagcact gatgttttac ttctttcacg ctacctttta 180
agagtaatcg acaagggagg aggcggctcg ggaggaggcg gctcgggagg aggcggctcg 240
catcatcatc atcatcatta a 261
<210>2
<211>282
<212>DNA
<213> Artificial sequence
<400>2
atggctcaat ataaagtcgg tgacttaaac ggtgacggag tggttaattc aactgacagt 60
gtaatattga aaagacatat aattaaattt tctgaaataa cagatccagt taaattgaaa 120
gctgctgatc ttaacggaga tggcaatata aactccagcg atgtttcatt aatgaagaga 180
tatctgctcc gtataataga taaatttccg gtagaaggag gaggcggctc gggaggaggc 240
ggctcgggag gaggcggctc gcatcatcat catcatcatt aa 282
<210>3
<211>86
<212>PRT
<213> Artificial sequence
<400>3
Met Val Leu Leu Gly Asp Val Asn Gly Asp Gly Thr Ile Asn Ser Thr
1 5 10 15
Asp Leu Thr Met Leu Lys Arg Ser Val Leu Arg Ala Ile Thr Leu Thr
20 25 30
Asp Asp Ala Lys Ala Arg Ala Asp Val Asp Lys Asn Gly Ser Ile Asn
35 40 45
Ser Thr Asp Val Leu Leu Leu Ser Arg Tyr Leu Leu Arg Val Ile Asp
50 55 60
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
His His His His His His
85
<210>4
<211>93
<212>PRT
<213> Artificial sequence
<400>4
Met Ala Gln Tyr Lys Val Gly Asp Leu Asn Gly Asp Gly Val Val Asn
15 10 15
Ser Thr Asp Ser Val Ile Leu Lys Arg His Ile Ile Lys Phe Ser Glu
20 25 30
Ile Thr Asp Pro Val Lys Leu Lys Ala Ala Asp Leu Asn Gly Asp Gly
35 40 45
Asn Ile Asn Ser Ser Asp Val Ser Leu Met Lys Arg Tyr Leu Leu Arg
50 55 60
Ile Ile Asp Lys Phe Pro Val Glu Gly Gly Gly Gly Ser Gly Gly Gly
65 70 75 80
Gly Ser Gly Gly Gly Gly Ser His His His His His His
85 90
<210>5
<211>261
<212>DNA
<213> Artificial sequence
<400>5
atggtactgc ttggcgatgt aaacggtgat ggaaccatta actcaactga cttgacaatg 60
ttaaagagaa gcgtgttgag ggcaatcacc cttaccgacg atgcaaaggc tagagcaaga 120
gttgacaaga atggatcgat aaacagcact gatgttttac ttctttcacg ctacctttta 180
agagtaatcg acaagggagg aggcggctcg ggaggaggcg gctcgggagg aggcggctcg 240
catcatcatc atcatcatta a 261
<210>6
<211>282
<212>DNA
<213> Artificial sequence
<400>6
atggctcaat ataaagtcgg tgacttaaac ggtgacggag tggttaattc aactgacagt 60
gtaatattga aaagacatat aattaaattt tctgaaataa cagatccagt taaattgaaa 120
gctgctagac ttaacggaga tggcaatata aactccagcg atgtttcatt aatgaagaga 180
tatctgctcc gtataataga taaatttccg gtagaaggag gaggcggctc gggaggaggc 240
ggctcgggag gaggcggctc gcatcatcat catcatcatt aa 282
<210>7
<211>86
<212>PRT
<213> Artificial sequence
<400>7
Met Val Leu Leu Gly Asp Val Asn Gly Asp Gly Thr Ile Asn Ser Thr
1 5 10 15
Asp Leu Thr Met Leu Lys Arg Ser Val Leu Arg Ala Ile Thr Leu Thr
20 25 30
Asp Asp Ala Lys Ala Arg Ala Arg Val Asp Lys Asn Gly Ser Ile Asn
35 40 45
Ser Thr Asp Val Leu Leu Leu Ser Arg Tyr Leu Leu Arg Val Ile Asp
50 55 60
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
His His His His His His
85
<210>8
<211>93
<212>PRT
<213> Artificial sequence
<400>8
Met Ala Gln Tyr Lys Val Gly Asp Leu Asn Gly Asp Gly Val Val Asn
1 5 10 15
Ser Thr Asp Ser Val Ile Leu Lys Arg His Ile Ile Lys Phe Ser Glu
20 25 30
Ile Thr Asp Pro Val Lys Leu Lys Ala Ala Arg Leu Asn Gly Asp Gly
35 40 45
Asn Ile Asn Ser Ser Asp Val Ser Leu Met Lys Arg Tyr Leu Leu Arg
50 55 60
Ile Ile Asp Lys Phe Pro Val Glu Gly Gly Gly Gly Ser Gly Gly Gly
65 70 75 80
Gly Ser Gly Gly Gly Gly Ser His His His His His His
85 90
<210>9
<211>516
<212>DNA
<213> Artificial sequence
<400>9
atgagcgacg gtgtggtggt ggaaattggc aaagtgaccg gtagcgtggg taccaccgtg 60
gaaattccgg tgtactttcg cggtgttccg agcaaaggta tcgccaactg cgattttgtt 120
ttccgctacg atccgaacgt gctggaaatc atcggcatcg atccgggtga catcatcgtg 180
gacccgaatc cgaccaagag cttcgatacc gccatttacc cggaccgcaa aatcattgtg 240
ttcctgttcg cagaggatag cggtaccggt gcctatgcca tcaccaaaga tggcgtgttc 300
gcaaagattc gcgcaaccgt gaaaagtagc gcaccgggct acattacctt tgacgaggtg 360
ggtggctttg ccgataacga tctggtggaa cagaaggtga gcttcattga cggtggcgtt 420
aacgtgggta atgccacacc gaccaagggt ggaggaggcg gctcgggagg aggcggctcg 480
ggaggaggcg gctcgcatca tcatcatcat cattaa 516
<210>10
<211>171
<212>PRT
<213> Artificial sequence
<400>10
Met Ser Asp Gly Val Val Val Glu Ile Gly Lys Val Thr Gly Ser Val
1 5 10 15
Gly Thr Thr Val Glu Ile Pro Val Tyr Phe Arg Gly Val Pro Ser Lys
20 25 30
Gly Ile Ala Asn Cys Asp Phe Val Phe Arg Tyr Asp Pro Asn Val Leu
35 40 45
Glu Ile Ile Gly Ile Asp Pro Gly Asp Ile Ile Val Asp Pro Asn Pro
50 55 60
Thr Lys Ser Phe Asp Thr Ala Ile Tyr Pro Asp Arg Lys Ile Ile Val
65 70 75 80
Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr Lys
85 90 95
Asp Gly Val Phe Ala Lys Ile Arg Ala Thr Val Lys Ser Ser Ala Pro
100 105 110
Gly Tyr Ile Thr Phe Asp Glu Val Gly Gly Phe Ala Asp Asn Asp Leu
115 120 125
Val Glu Gln Lys Val Ser Phe Ile Asp Gly Gly Val Asn Val Gly Asn
130 135 140
Ala Thr Pro Thr Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Gly Gly Gly Gly Ser His His His His His His
165 170
<210>11
<211>717
<212>DNA
<213> Artificial sequence
<400>11
atgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180
gtgaccacct tcggctacgg cctgcaatgc ttcgcccgct accccgacca catgaagctg 240
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300
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>12
<211>238
<212>PRT
<213> Artificial sequence
<400>12
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 230235
<210>13
<211>35
<212>DNA
<213> Artificial sequence
<400>13
caaaggctag agcaagagtt gacaagaatg gatcg 35
<210>14
<211>35
<212>DNA
<213> Artificial sequence
<400>14
ccattcttgt caactcttgc tctagccttt gcatc 35
<210>15
<211>35
<212>DNA
<213> Artificial sequence
<400>15
aattgaaagc tgctagactt aacggagatg gcaat 35
<210>16
<211>35
<212>DNA
<213> Artificial sequence
<400>16
ccatctccgt taagtctagc agctttcaat ttaac 35
<210>17
<211>5528
<212>DNA
<213> Artificial sequence
<400>17
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 atggtactgc ttggcgatgt aaacggtgat 5100
ggaaccatta actcaactga cttgacaatg ttaaagagaa gcgtgttgag ggcaatcacc 5160
cttaccgacg atgcaaaggc tagagcagac gttgacaaga atggatcgat aaacagcact 5220
gatgttttac ttctttcacg ctacctttta agagtaatcg acaagggagg 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
<210>18
<211>5549
<212>DNA
<213> Artificial sequence
<400>18
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 atggctcaat ataaagtcgg tgacttaaac 5100
ggtgacggag tggttaattc aactgacagt gtaatattga aaagacatat aattaaattt 5160
tctgaaataa cagatccagt taaattgaaa gctgctgatc ttaacggaga tggcaatata 5220
aactccagcg atgtttcatt aatgaagaga tatctgctcc gtataataga taaatttccg 5280
gtagaaggag gaggcggctc gggaggaggc ggctcgggag gaggcggctc gcatcatcat 5340
catcatcatt aagaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca 5400
ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 5460
caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 5520
tttgctgaaa ggaggaacta tatccggat 5549

Claims (10)

1. A mutant of fibrosome dockerin suitable for low calcium ion concentration, wherein D in dockerin is1D/N3D/N5D/N9D12D in amino acid sequence1The mutation of D to R of the site, i.e. to R1D/N3D/N5D/N9D12An amino acid sequence.
2. A mutant of fibrosome docking protein suitable for low calcium ion concentration is characterized in that the nucleotide sequence of fibrosome docking protein is shown as SEQ ID NO.1, and site-directed mutagenesis is carried out to obtain the mutant of the docking protein with the nucleotide sequence shown as SEQ ID NO. 5; or the nucleotide sequence of the fibrosome docking protein is shown as SEQ ID NO.2, and the nucleotide sequence of the obtained docking protein mutant is shown as SEQ ID NO.6 by site-specific mutagenesis.
3. A mutant of fibrosome docking protein suitable for low calcium ion concentration is characterized in that the amino acid sequence of the fibrosome docking protein is shown as SEQ ID No.3, and site-directed mutagenesis is carried out to obtain a mutant of the docking protein, wherein the amino acid sequence is shown as SEQ ID No. 7; or the amino acid sequence of the fibrosome docking protein is shown as SEQ ID NO4, and the mutant of the docking protein is obtained by site-directed mutagenesis, wherein the amino acid sequence is shown as SEQ ID NO. 8.
4. A method for preparing a mutant dockerin protein according to any one of claims 1-3, wherein D in the dockerin protein is used1D/N3D/N5D/N9D12Amino acid sequence based according to D1D mutation of the site is R, a site-directed mutation primer is designed, a pET28a (+) vector carrying a cellosome docking protein gene is used as a template, PCR is carried out, recombinant mutant plasmids are constructed, the mutant plasmids are converted into escherichia coli BL21(DE3), positive clones are selected for fermentation, thalli are collected after fermentation is finished, and the thalli are crushed and purified to obtain the cellosome docking protein mutant.
5. The method according to claim 4, wherein the strain is collected by centrifugation after fermentation, and the strain is subjected to ultrasonication and affinity chromatography to obtain the mutant of the fibronectin.
6. The method according to claim 4, wherein primer design for dockerin mutants was performed using dockerin DocA (vector DocA-pET28a) or DocB (vector DocB-pET28a) ligated between the NcoI and EcoRI cleavage sites of pET28a (+) vector, respectively, as a template, and PCR was performed to construct corresponding mutant plasmids; wherein the dockerin DocA is derived from Clostridium thermocellum (Clostridium thermocellum) xynY gene (GenBank: X83269.1), the DocB is derived from Clostridium thermocellum (Clostridium thermocellum) celQ gene (GenBank: AB047845.2), the nucleotide sequence of the DocA is shown as SEQ ID NO.1, and the nucleotide sequence of the corresponding mutant D-RA is shown as SEQ ID NO. 5; or the nucleotide sequence of DocB is shown as SEQ ID NO. 2; the nucleotide sequence of the corresponding mutant D-RB is shown as SEQ ID NO. 6; transforming the mutant plasmid into escherichia coli BL21(DE3), selecting positive clone for fermentation, collecting thalli after fermentation is finished, crushing the thalli, and purifying to obtain a fibrosome docking protein mutant;
preferably, in the preparation method, the dockerin DocA amino acid sequence is mutated into a mutant D-RA as shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO. 7; or the dockerin DocB amino acid sequence is mutated into a mutant D-RB as shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO. 8.
7. The method of claim 6, wherein the nucleotide sequence of the PCR amplification primer of the DocA dockerin mutant is as follows:
D-RA-F1 is shown as SEQ ID NO. 13;
D-RA-R1 is shown as SEQ ID NO. 14;
the nucleotide sequence of the PCR amplification primer of the dockerin DocB mutant is as follows:
D-RB-F1 is shown as SEQ ID NO. 15;
D-RB-R1 is shown in SEQ ID NO. 16.
8. The method according to claim 6, wherein the PCR reaction system:
1 μ L of plasmid vector template, 2 μ L of forward mutation primer F, 2 μ L of reverse mutation primer R, 10 μ L of 5 XFastmutation buffer, 1 μ L of Fastmutation DNA Polymerase, ddH2O34μL；
Preferably, in the preparation method, the PCR reaction conditions are as follows:
pre-denaturation at 95 ℃ for 2 min; 94 ℃ for 20sec, 55 ℃ for 10sec, 68 ℃ for 3min, 18 cycles; supplementary extension at 68 ℃ for 5 min;
preferably, after the PCR reaction is finished, the PCR amplification product needs to be digested by DnpI enzyme, and after the digestion reaction system is uniformly mixed, the digestion reaction system is digested for 1h at 37 ℃ to obtain a vector to be transformed;
preferably, the digestion system:
49 mu L of PCR amplification product and 1 mu L of DnpI enzyme;
preferably, the above-mentioned vector to be transformed is transformed into E.coli BL21(DE3) cells, and the transformant is plated to a medium containing kanamycin (50. mu.g.mL)-1) LB solid medium ofAfter overnight culture at 37 ℃, selecting single colonies, carrying out sequencing verification, and screening positive mutants to obtain recombinant escherichia coli D-RA-BL21 and D-RB-BL21 containing the genes of the fibrosome docking protein mutant D-RA and D-RB, respectively;
preferably, the correctly verified strains are inoculated to a strain containing kanamycin (50. mu.g.mL)-1) The cultured cells were cultured overnight at 37 ℃ in the liquid LB medium, and then transferred to the liquid LB medium and cultured to OD at 37 ℃600When the concentration is approximately equal to 1, the mixture is added until the final concentration is 1 mu m.mL-1The IPTG was induced at 26 ℃ for 8 hours at 200rpm, the induced cells were collected, resuspended in 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 with PBS-EP + buffer to obtain purified mutant cellulosome-dockerin D-RA and D-RB, respectively.
9. Use of a mutant of the cellulosome dockerin described in any one of claims 1-3 to interact with fibronectin to construct a protein complex.
10. Use according to claim 9, wherein the mutant dockerin interacts with mucin at low calcium ion concentrations to construct a protein complex;
preferably, the low calcium ion concentration is 10-7M-10-3M；
Preferably, the low calcium ion concentration is 10-7M-10-4M；
Preferably, the low calcium ion concentration is 10-7M-10-6M；
Preferably, the use of said dockerin mutant for intracellular interaction with fibronectin to construct a protein complex.
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