CsgA protein recombinant with excellent capacity of adsorbing microplastics in water and application of CsgA protein recombinant

Abstract

The invention discloses a CsgA protein recombinant with excellent capacity of adsorbing microplastics in water and application of the CsgA protein recombinant, and belongs to the technical field of gene engineering and protein engineering. The CsgA protein recombinant 1L7 is constructed and screened by carrying out site-directed mutagenesis on CsgA protein by adopting PCR (Polymerase Chain Reaction) amplification and then connecting a hydrophobic short peptide CBM1 through a connecting peptide L7. When the pH value is 7, the removal efficiency of the 1L7 recombinant protein to PS is 91.57%, the removal efficiency of the 1L7 recombinant protein to PET can reach 92.37%, and the protein has high adsorption capacity to micro-plastics in a water body environment with the pH value being 3-10 and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to a CsgA protein recombinant with excellent ability to absorb microplastics in water and application thereof, belonging to the technical field of genetic engineering and protein engineering. Background Art

[0002] Microplastics are usually defined as those with a diameter of less than 5mm. Microplastics pose potential health risks to the human body due to their small particle size, large number, and adsorption properties. They can destroy the balance of intestinal flora in the intestines, easily leading to malnutrition, developmental delay, etc. Nano-microplastics are cytotoxic and can inhibit cell division. Microplastics are also a potential way to transfer toxic organic compounds and heavy metals to aquatic organisms. Although the current treatment processes of water plants at home and abroad can remove a large proportion of microplastics, a large amount of small-particle microplastics still remain in the water. Therefore, it is urgent to strengthen conventional processes and develop emerging technologies to remove microplastics in water.

[0003] CsgA nanofibers derived from the protein matrix of Escherichia coli biofilm are a new type of amyloid protein functional material with great potential. First, the protein has a characteristic β-folded structure, so it has excellent mechanical properties and excellent environmental tolerance unique to amyloid protein; secondly, genetic engineering can be used to achieve rational regulation of the molecular level function of CsgA protein, and the fusion protein can still self-assemble to form nanofibers, and retain the function of the fusion protein. The present invention obtains a mutant strain with good microplastic removal ability by performing site-directed mutagenesis on the parent CsgA protein. Summary of the invention

[0004] The purpose of the present invention is to provide a CsgA protein recombinant with good ability to adsorb microplastics in water and its application.

[0005] The present invention provides a recombinant CsgA protein, wherein the recombinant CsgA protein is based on a parent CsgA protein and has one or more of the following mutations:

[0006] (1) Aspartic acid at position 127 was mutated to arginine;

[0007] (2) glycine at position 108 mutated to tyrosine;

[0008] (3) Lysine at position 109 was mutated to tryptophan.

[0009] In one embodiment, the parent CsgA protein has the amino acid sequence shown in SEQ ID NO.1.

[0010] In one embodiment, the recombinant CsgA protein is further connected to a hydrophobic short peptide CBM1 at the C-terminus via a connecting peptide.

[0011] In one embodiment, the amino acid sequence of the connecting peptide is shown as SEQ ID NO.5; the nucleotide sequence encoding the connecting peptide is shown as SEQ ID NO.6.

[0012] In one embodiment, the amino acid sequence of the hydrophobic short peptide CBM1 is shown as SEQ ID NO.7; the nucleotide sequence encoding the hydrophobic short peptide CBM1 is shown as SEQ ID NO.8.

[0013] The present invention also provides a gene encoding the recombinant CsgA protein.

[0014] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.4.

[0015] The invention also provides a recombinant vector carrying the gene.

[0016] In one embodiment of the present invention, the recombinant vector uses pET-28a as an expression vector.

[0017] The present invention also provides a recombinant cell expressing the above-mentioned CsgA protein recombinant 1L7, or carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

[0018] In one embodiment, the recombinant cell uses bacteria or fungi as host cells.

[0019] In one embodiment, the host cell is Escherichia coli BL21 (DE3).

[0020] In one embodiment, the recombinant cell uses Escherichia coli BL21 (DE3) as a host and pET28a as a vector to express the recombinant CsgA protein.

[0021] The present invention also provides a method for treating microplastics in water, wherein the method uses the CsgA protein recombinant 1L7 to adsorb microplastics in water.

[0022] In one embodiment, the microplastics include but are not limited to PS or PET.

[0023] In one embodiment, the microplastics include but are not limited to PS particles or PET particles with a particle size of ≤500 nm.

[0024] In one embodiment, the pH of the water body is 3-10.

[0025] Beneficial Effects

[0026] The present invention provides a CsgA protein recombinant 1L7, which can achieve a removal rate of 91.57% for 500nm PS in water with a pH value of 7 at 28h, and a removal efficiency of 92.37% for PET. Therefore, this recombinant can greatly improve the application value of CsgA amyloid nanofibril protein in water treatment. And as a green biomaterial, CsgA protein is safer during application, so the CsgA protein recombinant 1L7 with good microplastic adsorption capacity provided by the present invention has high application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The removal efficiency of 1L7 protein for 0.2 mg / mL PS at different pH values.

[0028] Figure 2 The removal efficiency of 1L7 protein for 0.2 mg / mL PET at different pH values.

[0029] Figure 3 Actual photo of 1L7 recombinant protein adsorbing 0.2 mg / mL PS / PET at pH 7 at room temperature 25°C. DETAILED DESCRIPTION

[0030] The E. coli JM109 involved in the following examples was purchased from Beina Biotechnology; the pET-28a plasmid involved in the following examples was purchased from Fenghui Biotechnology; the plasmid pET28a-CsgA noSS involved in the following examples was purchased from Fenghui Biotechnology TVNFKLY-HisTag and E. coli BL21(DE3) / pET28a-CsgA noSS TVNFKLY-HisTag Published in the master's thesis "Biological Removal of Heavy Metal Ions Based on Functional Modification of CsgA Protein".

[0031] The culture medium involved in the following examples is as follows:

[0032] LB liquid medium: yeast powder 5g·L -1 , Tryptone 10 g·L -1 、NaCl 10g·L -1 .

[0033] LB solid medium: yeast powder 5g L -1 , Tryptone 10 g·L -1 、NaCl 10g·L -1 , agar powder 20g·L -1 .

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Microplastic removal capacity determination method:

[0036] First, prepare a 0.2 mg / mL PS / PET suspension, and then add the purified 1L7 protein material to the PS / PET suspension at a final concentration of 60 μg / mL. Then mix at 300 rpm for 5 minutes, and then let it stand to wait for the 1L7 protein material to adsorb PS / PET and then settle.

[0037] Calculation of removal rate:

[0038]

[0039] Where A is the removal efficiency of PS / PET (%), C1 is the PS / PET concentration after adsorption, and C2 is the initial PS / PET concentration.

[0040] Example 1: Construction of recombinant CsgA protein

[0041] (1) Construction of recombinants

[0042] The CsgA protein with an amino acid sequence as shown in SEQ ID NO.1 was used as the parent protein, aspartic acid at position 127, glycine at position 108 and lysine at position 109 were selected for site-directed mutagenesis, and a hydrophobic short peptide CBM1 was connected to its C-terminus via a linker peptide.

[0043] According to the sequence of CsgA protein (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1), primers for mutation were designed (as shown in Table 1) to clone the plasmid pET28a-CsgAnoSS TVNFKLY-HisTag The CsgA protein gene was subjected to site-directed mutagenesis.

[0044] Table 1 Primers

[0045]

[0046]

[0047] The PCR reaction system was as follows: PrimeSTAR Max Premix (2×) 25 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, template DNA 1 μL, and double distilled water was added to 50 μL;

[0048] The amplification conditions of PCR products were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 2 min, 30 cycles; and finally incubation at 72°C for 10 min.

[0049] The PCR amplification product was detected by 1% agarose gel electrophoresis. After confirming that the amplification product was of the correct size, 0.5 μL of methylated template digestion enzyme (DpnI) was added to 10 μL of the amplification product, mixed, and reacted at 37°C for 1 h. The amplification product treated with DpnI was transformed into Escherichia coli JM109 competent cells, and the transformation product was coated on a plate with kanamycin (final concentration 50 mg·L -1 ) on LB solid medium and cultured at 37°C for 8-10 h. The single colony grown on the plate was inoculated into a LB solid medium supplemented with kanamycin (final concentration 50 mg·L -1 ) in LB liquid culture medium at 37°C and 220 r / min for 8 to 10 h, and then the plasmid was extracted to obtain a mixed recombinant plasmid containing different mutant genes: pET28a-recombinant.

[0050] Example 2: Construction of recombinant bacteria and expression of recombinant CsgA protein (1L7 protein)

[0051] (1) The recombinant plasmid pET28a-recombinant obtained in Example 1 was transformed into competent Escherichia coli BL21 (DE3), and the transformation products were respectively applied to LB solid medium containing kanamycin resistance, and cultured at 37° C. for 10 to 12 hours. Then, the single colonies grown on the plate medium were inoculated into LB liquid medium containing kanamycin, and cultured at 37° C. and 220 rpm for 10 to 12 hours to prepare seed solutions;

[0052] (2) The seed solution obtained in step (1) was transferred to LB liquid culture medium at a ratio of 5% (v / v), cultured at 37°C and 220 rpm for 2 h, and then an induction solution IPTG with a final concentration of 0.5 mM was added at 1‰ (v / v), and cultured at 30°C and 220 rpm for 8 h to obtain a fermentation broth.

[0053] (3) The fermentation broth prepared in step (2) was centrifuged at 4°C and 5000 rpm for 10 min to obtain a fermentation precipitate (bacteria).

[0054] (4) Dissolve the resulting bacteria in guanidine hydrochloride solution at a ratio of 5 g of bacteria to 50 ml of 8 M guanidine hydrochloride solution (adjust the pH value of the solution to 7.4), stir at room temperature and 300 r / min for 12 h, centrifuge the dissolved product at 10,000 r / min for 1 h, collect the supernatant, pass it through a 0.45 μm water membrane, and place it in a 4°C refrigerator for use.

[0055] (5) The protein was purified by nickel ion affinity chromatography using a Ni-NTA gravity chromatography column, washed with 20 mM imidazole, and eluted with 200 mM imidazole to obtain the target protein. The collected protein solution was tested for purity by SDS-PAGE gel electrophoresis, and the concentration was tested by a Bradford protein concentration assay kit.

[0056] Example 3: Effect of 1L7 protein on the removal of PS and PET

[0057] The protein prepared in Example 2 was used to adsorb microplastics. A total reaction system of 1.5 mL was prepared, 1 L7 protein concentration was 60 mg / L, PS with a particle size of 500 nm was 0.1 mg / L, pH was adjusted to 3-10, and the mixture was shaken and mixed at 25 °C for 10 min, then allowed to stand for 28 h, and the removal rate was measured after the adsorption of microplastics and precipitation. E. coli BL21 (DE3) / pET28a-CsgA noSS was used according to the method of Examples 1-2. TVNFKLY-HisTag The parent CsgA protein was prepared and the parent CsgA protein was used as a control. The results are shown in Table 2 and Figure 1 shown.

[0058] The results showed that compared with CsgA protein, 1L7 protein had good PS removal ability in a wide range of pH 3 to 10, among which the PS removal efficiency was 91.57% at pH 7.

[0059] Table 2 Removal efficiency of PS / PET by 1L7 protein at different pH

[0060]

[0061] Comparative Example 1: Electrostatic Interaction Module Modification

[0062] The specific implementation method is the same as Example 1, except that the modification was performed to improve the isoelectric point, and eight sites of CsgA, including D67, D80, D86, D87, D91, D104, E112 and D127, were mutated to construct the mutants shown in Table 3 (without fusion of hydrophobic short peptides). The expression and purification were performed according to the method of Example 2. Microplastics were adsorbed in a reaction system of pH = 7 according to the method of Example 3, and the results are shown in Table 3.

[0063] Table 3 PS removal efficiency of different mutants of electrostatic interaction modification module

[0064]

[0065]

[0066] As shown in Table 3, the four mutants, D127R, D104R / E112R, D67R / D80R / D86R / D87R / D91R and D80R / D86R / D87R, had significantly improved PS removal abilities compared with CsgA, among which D127R had the best effect, with a PS removal efficiency of 89.5% at 60 h.

[0067] Comparative Example 2: π-π interaction module modification

[0068] The specific implementation method is the same as Example 1, except that, in order to enhance the π-π interaction, G108 and K109 were mutated on the basis of the mutant D127R, and the mutants shown in Table 4 (without fusion of hydrophobic short peptide) were constructed. Expression and purification were performed according to the method of Example 2. Microplastics were adsorbed in an environment of pH=7 according to the method of Example 3, and the results are shown in Table 4.

[0069] Table 4 Removal efficiency of PS by mutants modified with π-π interaction modules

[0070]

[0071] As shown in Table 4, the mutant D127R / G108Y / K109W had the best PS removal effect compared with the mutant D127R, and the removal efficiency could reach 93.57% at 48 h.

[0072] Comparative Example 3: Fusion Protein Module

[0073] The specific implementation method is the same as Example 1, except that, based on Comparative Example 2, the mutants shown in Table 5 were constructed for the modification of hydrophobic interaction fusion hydrophobic short peptides, wherein 1L1 to 1L8 respectively represent the connection of hydrophobic short peptide CBM1 at the C-terminus of the D127R / G108Y / K109W mutants through Linker 1 to Linker 8; 2L1 to 2L8 represent the connection of hydrophobic short peptide CBM2 at the C-terminus of the mutant D127R / G108Y / K109W through Linker 1 to Linker 8;

[0074] 3L1~3L8 indicates that the hydrophobic short peptide CBM3 is connected to the C-terminus of the mutant D127R / G108Y / K109W through linker1~linker8; 4L1~4L8 indicates that the hydrophobic short peptide CBM4 is connected to the C-terminus of the mutant D127R / G108Y / K109W through linker1~linker8; the sequence of the hydrophobic short peptide CBM1~CBM4 is shown in Table 6, and the sequence of linker1~linker8 is shown in Table 7.

[0075] The expression and purification were performed according to the method of Example 2. Microplastics were adsorbed under the environment of pH=7 according to the method of Example 3. The results are shown in Table 5.

[0076] Table 5 Removal efficiency of PS by fusion protein module mutants

[0077]

[0078]

[0079] Table 6 Hydrophobic short peptides CBM1-4

[0080]

[0081] Table 7 Linker 1 to linker 8 and their sequences

[0082]

[0083] As shown in Table 5, the adsorption efficiency of PS by the six mutants, 1L1, 1L4, 1L7, 2L2, 2L3 and 4L3, reached more than 92% at 24 h, with 1L7 having the highest efficiency of 93.75%.

[0084] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant CsgA protein, characterized in that: Based on the parent CsgA protein, it has one or more of the following mutations: (1) Aspartic acid at position 127 mutated to arginine, (2) glycine at position 108 mutated to tyrosine; (3) Lysine at position 109 was mutated to tryptophan.

2. The recombinant CsgA protein according to claim 1, characterized in that The recombinant CsgA protein is also connected to a hydrophobic short peptide CBM1 at the C-terminus via a connecting peptide.

3. The recombinant CsgA protein according to claim 1 or 2, characterized in that: The amino acid sequence of the connecting peptide is shown in SEQ ID NO.5; the amino acid sequence of the hydrophobic short peptide CBM1 is shown in SEQ ID NO.

7.

4. A gene encoding the recombinant CsgA protein according to any one of claims 1 to 3.

5. A recombinant vector carrying the gene according to claim 4.

6. A recombinant microbial cell expressing the recombinant CsgA protein according to any one of claims 1 to 3, or carrying the gene according to claim 4, or containing the recombinant vector according to claim 5.

7. A recombinant Escherichia coli, characterized in that E. coli BL21 (DE3) is used as the expression host, and pET28a is used as the vector to express the CsgA protein recombinant according to any one of claims 1 to 3.

8. A method for treating microplastics in water, characterized in that: The recombinant CsgA protein according to any one of claims 1 to 3 is contacted with microplastics in water; the microplastics include but are not limited to PS or PET.

9. A method for improving the adsorption capacity of CsgA protein on microplastics in water, characterized in that: The glycine at position 108 of the amino acid sequence of the CsgA protein was mutated to tyrosine, the lysine at position 109 was mutated to tryptophan, and the aspartic acid at position 104 was mutated to arginine, and a hydrophobic short peptide CBM1 was connected to the C-terminus of the protein.

10. Use of the recombinant CsgA protein according to any one of claims 1 to 3, or the recombinant microbial cell according to claim 6, or the recombinant Escherichia coli according to claim 7, or the method according to claim 8 in treating PS and / or PET in water.

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