Cellulosome docking protein mutant suitable for low calcium ion concentration and application
阅读说明：本技术 一种适用于低钙离子浓度的纤维小体对接蛋白突变体及应用 (Cellulosome docking protein mutant suitable for low calcium ion concentration and application ) 是由 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 于 2020-07-27 设计创作，主要内容包括：本发明公开了一种适用于低钙离子浓度的纤维小体对接蛋白突变体及应用,一种适用于低钙离子浓度的纤维小体对接蛋白突变体为对接蛋白中的D1D/N 3D/N 5D/N9D12氨基酸序列中的D1位点的D突变为R,即突变为R1D/N 3D/N 5D/N9D12氨基酸序列；所述对接蛋白突变体适用于低钙离子浓度为10<Sup>-7</Sup>M-10<Sup>-4</Sup>M的条件与黏连蛋白相互作用；进一步的适用于低钙离子浓度为10<Sup>-7M</Sup>-10<Sup>-6</Sup>M的条件与黏连蛋白相互作用；适用于细胞内环境中与黏连蛋白相互作用；解决了现有纤维小体元件在低钙离子浓度下无法自组装的问题。(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 ‑7 M‑10 ‑4 The condition of M interacts with mucin; further suitable for low calcium ion concentration of 10 ‑7M ‑10 ‑6 The 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.)
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.
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.
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.