CN110423738B

CN110423738B - Method for separating and purifying beta-glucosidase fusion protein

Info

Publication number: CN110423738B
Application number: CN201910702477.7A
Authority: CN
Inventors: 韩娟; 王赟; 王蕾; 李春梅; 和兴晨; 张红清; 旦增曲珍; 倪良
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2022-10-28
Anticipated expiration: 2039-07-31
Also published as: CN110423738A

Abstract

The invention mainly relates to a method for separating and purifying beta-glucosidase fusion protein, belonging to the technical field of separation and purification; the invention firstly induces and expresses the fusion protein GLEGB, and then bacterial liquid containing the fusion protein, surfactant and (NH 4) ₂ SO ₄ Mixing to serve as a lower phase, taking an upper phase as PEG2000, performing flotation, respectively collecting upper and lower phase solutions after flotation, recording the volume, and measuring the enzyme activity and the protein content in the upper phase and the lower phase after flotation; adding ammonium sulfate into the collected upper phase mixed solution, heating in water bath, and centrifuging to remove supernatant; then, resuspending the precipitate by using a Tris-HCl buffer solution, incubating for a certain time at a low temperature, and centrifuging to remove impurities to obtain a purified target enzyme solution; the invention combines ATPF and ITC to purify the fusion enzyme GLEGB in two steps, greatly improves the purification efficiency, and has simple operation, low cost and no pollution.

Description

Method for separating and purifying beta-glucosidase fusion protein

Technical Field

The invention mainly relates to a method for separating and purifying beta-glucosidase fusion protein, in particular to a method for separating and purifying the fusion beta-glucosidase by constructing a flotation system by using the performance of a hydrophobic label and combining a temperature sensitive material; belongs to the technical field of separation and purification.

Background

Beta-glucosidase (beta-Glu) is a hydrolase that can hydrolyze beta-D-glucosidic bonds bound to non-reducing ends, and has a close relationship with terpene-based aroma precursors, and therefore has important application values in the fields of cellulose degradation, food flavor improvement and the like. In addition, it is also involved in sugar metabolism of organisms and plays an important role in maintaining normal physiological functions of the organisms. Therefore, the research on the beta-Glu has important theoretical and practical value.

In the enzymolysis process of cellulose, the beta-glucosidase can be applied to the milk product industry to decompose lactose, and can be used as a flavor enzyme to be applied to (fruit) wine, tea and fruit juice to play a role in enhancing the flavor; the application of beta-glucosidase activity in medicine also plays an important role in the information acquisition of certain diseases; in daily chemical industry, the transglycosylation of beta-glucosidase is utilized to produce alkyl glycoside of nonionic surfactant. Therefore, the separation and purification of beta-glucosidase (beta-Glu) has very important practical significance.

The currently used methods for purifying beta-glucosidase (beta-Glu) mainly comprise: ammonium sulfate/acetone fractional precipitation, DEAE anion exchange chromatography, gel filtration, sephadex column chromatography, dialysis, high performance liquid chromatography, etc. The defects of the methods are that a single method can not complete the separation and purification of beta-glucosidase (beta-Glu), a plurality of methods are needed to be combined, the operation process is complex, the cost is increased, the workload is large, the required equipment is expensive, the yield is low, and the purification multiple is low, so that the method has certain limitations.

Disclosure of Invention

The invention aims to overcome the defects of complex extraction process, low separation and purification efficiency, incapability of recycling selected materials and the like in the conventional method for separating and purifying the protein, and provides a novel method for separating and purifying the protein by combining aqueous two-phase flotation (ATPF) with reverse switching cycle (ITC).

In order to achieve the technical purpose, the technical means adopted by the invention are as follows:

(1) Inducible expression of fusion protein GLEGB

Coding and designing an ELP amino acid sequence and GB polypeptide, connecting a connecting peptide linker to glucosidase (Glu) to construct a recombinant plasmid pET-GLEGB (the GLEGB is the abbreviation of Glu-linker-ELP-GB); the induced expression of the recombinant plasmid was carried out in E.coli BL21 cells, and a monoclonal of the objective transformant was first picked up and cultured overnight in the presence of kanamycin. Then, the scale-up culture was carried out at a ratio of 1%. The cells were kept on ice and an inducer of isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added and the culture was continued overnight. And (4) centrifuging and collecting bacterial precipitates. The pellet was washed with Tris-HCl buffer and resuspended. After adding phenylmethylsulfonyl fluoride (PMSF), the mixture was crushed in an ice-water bath using a sonicator.

(2) Separation and purification of fusion protein GLEGB by ATPF

Mixing bacterial liquid containing fusion protein, surfactant and (NH) ₄ ) ₂ SO ₄ Mixing to serve as a lower phase, taking an upper phase as PEG2000, performing flotation, collecting upper phase enzyme liquid after flotation, accurately recording the volume, and determining the recovery rate of enzyme activity and a purification factor after flotation.

Wherein: the concentration of ammonium sulfate in the lower phase is 0.25g/mL-0.45g/mL;

the concentration of the surfactant is 1.0% -2.0%, wherein the surfactant is BS-12 (dodecyl dimethyl betaine);

wherein the volume ratio of the lower phase to the upper phase is 5:1; wherein the bacterial liquid dosage of the fusion protein contained in the lower phase accounts for 1/5 of the total volume of the lower phase.

The concentration of PEG2000 in the upper phase is 30-50%.

The flotation condition is about 20-40min under the condition of 20-40mL/min of flow rate.

(3) Separation and purification of fusion protein GLEGB by combining ATPF with ITC

Adding a certain amount of ammonium sulfate into the upper phase enzyme solution collected in the step (2) for ITC (reverse conversion cycle) purification, heating in a water bath, centrifuging and discarding the supernatant. Resuspending the pellet with Tris-HCl buffer, incubating at low temperature for a certain period of time, and centrifuging to remove the pellet. The supernatant is the purified target enzyme solution.

Wherein: ammonium sulfate was added to the upper enzyme solution so that the final concentration of ammonium sulfate was 0.1-1M.

The heating time in water bath is 10min, and the temperature is 15-37 ℃.

The two centrifugation conditions are as follows: 12000rpm,15min.

Tris-HCl buffer: it is pre-cooled, 50mM, pH8.0 before use.

Incubation conditions of the resuspended solution: 4 ℃ for 10-40min.

In the invention, in aqueous two-phase flotation (ATPF), GLEGB realizes the purpose of floating a target enzyme from a lower phase to an upper phase by depending on the hydrophobic effect and bubble adsorption of a GB label, and in order to prove the hydrophobic effect of the GB label, compared with the flotation effects of a plurality of fusion proteins (Glu, GLE and GLG), the addition of the GB label greatly improves the flotation effect relative to Glu without the label, and the hydrophobic effect of the GB label on the fusion protein is further verified.

The GB and ELP labels are fused with target enzyme, and the fusion protein is purified by utilizing the hydrophobic effect of the GB label and the temperature-sensitive characteristic of the ELP label. The isolated and purified GLEGB is compared with the purchased Glu by UV-vis, FT-IR and CD characterization, and is found to cause no damage to the structure of the enzyme in the purification process.

The beneficial effects of the invention are as follows:

in the invention, a fusion enzyme GLEGB (Glu-Linker-ELP-GB) is used, namely a binary tag, elastin polypeptide (ELP) and graphene binding peptide (GB) are added into beta-Glu for separating and purifying the beta-Glu fusion enzyme. ELP is a synthetic peptide-based polymer, a thermo-responsive polypeptide, that reversibly aggregates above a critical temperature, referred to as the phase transition temperature (Tt). And when the temperature is lower than Tt, ELP becomes soluble again. The recombinant ELP fusion proteins will continue to maintain the reversible phase transition properties of ELP, which are used in protein purification, known as the reverse switching cycle (ITC). The graphene binding peptide (GB) His-Asn-Trp-Tyr-His-Trp-Trp-Pro-His has strong affinity on the surface of the graphene binding peptide (GB) because the graphene binding peptide is rich in hydrophobic aromatic amino acid residues, so that bubbles can be adsorbed, and the graphene binding peptide can be used for separating and purifying target substances with poor hydrophobicity or surface activity, thereby improving the purification rate in the ATPF purified protein.

(1) The invention uses the fusion enzyme in the separation and purification of the target enzyme by the double-aqueous phase flotation for the first time, and the double-aqueous phase flotation (ATPF) method has the advantages of simple equipment, high selectivity, high separation efficiency, simple operation, mild operation condition, easy amplification and the like. The specific label is used for separating and purifying the enzyme, so that the specificity is high, the separation and purification efficiency is greatly improved, and finally the enzyme activity recovery rate reaches 124.92%.

(2) The separation and purification of the fusion enzyme by utilizing the hydrophobicity of the GB label is a big highlight of the invention, and the GB label can be attached with the fusion enzyme to enable the fusion protein to enter the upper phase along with bubbles by being adsorbed on the surface of the bubbles generated when nitrogen is introduced, thereby achieving the purification purpose.

(3) The invention combines ATPF and ITC to purify the fusion enzyme GLEGB in two steps, thereby greatly improving the purification efficiency and finally obtaining the enzyme activity recovery yield Ye _b Up to 124.92% + -0.0083 and purification factor PF _b 24.26 +/-0.0220 is achieved. And the whole operation is simple, the cost is low, and no pollution is caused.

(4) The invention not only realizes the separation of the polymers in the upper phase, but also effectively solves the limitation of other separation means at present, and in addition, the invention also realizes the recycling of the PEG2000 obtained by separation, thereby saving the cost, protecting the environment and having great practical significance.

Drawings

FIG. 1 is a schematic structural diagram of recombinant plasmid pET-GLEGB (containing ELP and GB tags).

FIG. 2 is a diagram of the phase change mechanism of ELP.

FIG. 3 is a SDS-PAGE gel of ATPF combined with ITC technology for purification of GLE, GLEH and GLEGB. Wherein, lane 1: crude enzyme solution of GLEGB; lane 2: purified GLEGB; lane M: a protein molecular weight marker; lane 3: GLE crude enzyme solution; lane 4: purified GLE; lane 5: crude enzyme solution of GLEH; lane 6: purified GLEH.

FIG. 4 shows the case of 6 cycles of PEG2000 in GLEGB purified by ATPF combined with ITC technology.

FIG. 5 is a comparison of UV spectra (FIG. 5 a), IR spectra (FIG. 5 b) and circular dichroism spectra (FIG. 5 c) of purchased pure Glu and purified GLEGB.

FIG. 6 shows the changes in the enzymatic activity of Glu in 2h in solutions of PEG 600 (FIG. 6 a), PEG2000 (FIG. 6 b), PEG 4000 (FIG. 6 c), PEG 8000 (FIG. 6 d), and PEG 10000 (FIG. 6 e).

FIG. 7 shows the enzymatic activity of Glu at K ₃ PO ₄ (FIG. 7 a), K ₃ C ₆ H ₅ O ₇ (FIG. 7 b), (NH) ₄ ) ₂ SO ₄ (FIG. 7 c) in solution.

FIG. 8 shows the changes in the enzymatic activities of Glu in the solutions of BS-12 (FIG. 8 a), CTAB (FIG. 8 b), SDS (FIG. 8 c), triton X-100 (FIG. 8 d).

FIG. 9 shows the Ye of GLEGB purified by separation of PEG molecular weight from ATPF _a And PF _a The influence of (c).

FIG. 10 shows the concentration of PEG2000 vs. Ye of GLEGB isolated and purified by ATPF _a And PF _a The influence of (c).

FIG. 11 is (NH) ₄ ) ₂ SO ₄ Concentration versus Ye of purified GLEGB isolated from ATPF _a And PF _a The influence of (c).

FIG. 12 is a graph of the concentration of BS-12 vs. Ye of isolated and purified GLEGB from ATPF _a And PF _a The influence of (c).

FIG. 13 is the Ye of GLEGB separated and purified by flotation flow rate vs. ATPF _a And PF _a The influence of (c).

FIG. 14 is the effect of flotation time on Yea and PFa for ATPF separation and purification of GLEGB

Detailed Description

In order to make the technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are further described below with reference to the accompanying drawings by way of examples, so that the technical solutions of the present invention are clearer and easier to understand.

Example 1:

(1) Inducible expression of the fusion protein GLEGB

Encoding design ELP (elastin-like polypeptides), represented as ELP (VPGVG) ₅₀ The recombinant plasmid pET-GLEGB (Glu-linker-ELP-GB) was constructed by inserting a GB (Graphene binding) polypeptide and linking it to glucosidase (Glu) using a linker. Recombinant plasmids pET-GLE (Glu-linker-ELP), pET-GLEH (Glu-linker-ELP-6 His) were additionally constructed, in which pET-GLG (Glu-linker-GB) and pET-GLH (Glu-linker-6 His) were used as controls. The recombinant plasmid was transformed into E.coli BL21 cells to express the fusion gene.

FIG. 1 is a schematic diagram of the structure of recombinant plasmid pET-GLEGB (containing ELP and GB label).

The amino acid sequence of the linker is shown in SEQ.ID.NO. 2; the amino acid sequence of the ELP is shown in SEQ.ID.NO. 3; the amino acid sequence of the GB is shown in SEQ.ID.NO. 4. Each amino acid sequence is as follows:

SEQ.ID.NO.1(Glu)：

MDDVDNDTLVTFPDDFKLGAATASYQIEGGWDADGKGPNIWDTLTHERPHLVVDRST GDVADDSYHLYLEDVRLLKDMGAEVYRFSISWARILPEGHDNNVNEAGIEYYNKLIDALLR NGIEPMVTMYHWDLPQKLQDLGGWPNRILAKYAENYARVLFSNFGDRVKQWLTFNEPLTF MDAYASDTGMAPSVDTPGIGDYLTAHTVILAHANIYRLYEREFREEQQGQVGIALNIHWCE PETGSPKDVEACERYQQFNLGIYAHPIFSENGDYPSVLKARVDANSASEGYTTSRLPKFTPE EVAFVNGTYDFLGLNFYTAVVGRDGVEGEPPSRYRDMGTITSQDPEWPESASSWLRVVPW GFRKELNWIANEYGNPPIFITENGFSDYGGVNDTNRVLYYTEHLKEMLKAIHIDGVNVIGYT AWSLIDNFEWLRGYTERFGIHAVNFIDPSRPRTPKESARVLTEIFKTRQIPERFRDAS

SEQ.ID.NO.2(linker)：GGGGSGGGGSGGGGSEL

SEQ.ID.NO.3(ELP)：

VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGV PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGKL

SEQ.ID.NO.4(GB)：HNWYHWWPH

induced expression of recombinant plasmids in E.coli BL21 cells: first, a monoclonal bacterium of the objective transformant was selected in the presence of kanamycin, inoculated into 3mL of LB liquid medium, and cultured overnight at 190rpm at 25 ℃. 2mL of the bacterial liquid is taken and transferred into 200mL of liquid LB culture medium containing the kanamycin according to the proportion of 1 percent for amplification culture until the OD600 value is 0.1. The suspension was allowed to stand on ice for 10min and IPTG was added to bring the final concentration to 0.1mM, and the culture was continued overnight at 190rpm at 20 ℃. Then, the cells were centrifuged at 4500rpm for 20min at 4 ℃ to remove the medium, and the pellet was collected. And washed two to three times with 50mM Tris-HCl buffer solution, pH8.0, and centrifuged under the same conditions to remove the buffer solution. The obtained pellet of the cells was resuspended in Tris-HCl buffer, PMSF was added to a final concentration of 1mM, and then disrupted in an ice-water bath using an ultrasonicator. The parameters of the ultrasonograph were set as: power 140W, crushing time 10s, interval time 10s and total working time 30min. After the disruption was completed, the disruption solution was centrifuged at 14000rpm for 15min at 4 ℃ to remove the precipitate, and the supernatant was collected into a new EP tube and stored at-80 ℃. If the obtained thallus is not broken immediately, the thallus needs to be stored in a refrigerator at the temperature of minus 80 ℃.

(2) Separation and purification of fusion protein GLEGB by ATPF

Taking a certain amount of bacterial liquid containing fusion protein, surfactant BS-12 and (NH) ₄ ) ₂ SO ₄ Adding into a 20mL colorimetric cylinder and dissolving with distilled water to the scale mark to make the concentration of the surfactant BS-12 be 2.0%, (NH) ₄ ) ₂ SO ₄ Shaking the solution uniformly to be transferred into a flotation tank as a lower phase, adding 30% PEG2000 as an upper phase, carrying out flotation, controlling the flow rate of the flotation at 20mL/min and carrying out the flotation for 40min, collecting upper phase enzyme liquid after bubbles in the flotation tank completely disappear after the flotation is finished, accurately recording the volume, and measuring the enzyme activity recovery rate Ye _a 92.52 percent, and a purification factor PF _a Up to 3.31. The experiments were all performed at room temperature throughout.

2mL of the supernatant enzyme solution was taken, and 0.026g of ammonium sulfate was added to the solution to adjust the concentration of ammonium sulfate in the enzyme solution to 0.1M, and dissolved. The mixture was then heated in a 15 ℃ water bath for 10min and centrifuged at 12000rpm for 15min to remove the supernatant. The pellet was resuspended in precooled Tris-HCl buffer (50mM, pH 8.0), and the resuspended solution was incubated at 4 ℃ for 10min, centrifuged again at 12000rpm for 15min and the pellet discarded. The supernatant is the final purified target enzyme solution. Determination of enzyme activity recovery Ye _a 120.19%, purifying factor PF _a Up to 13.74.

Example 2:

(1) Inducible expression of fusion protein GLEGB

Recombinant plasmids pET-GLEGB, pET-GLE and pET-GLEH were constructed in accordance with the procedure of step (1) in example 1. The recombinant plasmid was transformed into E.coli BL21 cells to express the fusion gene.

The induced expression of the recombinant plasmid was carried out in E.coli BL21 cells. First, a monoclonal bacterium of the objective transformant was selected in the presence of kanamycin, inoculated into 3mL of LB liquid medium, and cultured overnight at 45 ℃ and 190 rpm. 2mL of the bacterial liquid is taken and transferred into 200mL of liquid LB culture medium containing the kanamycin according to the proportion of 1 percent for amplification culture until the OD600 value is 0.8. The suspension was allowed to stand on ice for 30min and IPTG was added to bring the final concentration to 0.5mM, and the culture was continued overnight at 37 ℃ and 190 rpm. Then, the cells were centrifuged at 4500rpm for 20min at 4 ℃ to remove the medium, and the pellet was collected. And washed two to three times with 50mM Tris-HCl buffer solution, pH8.0, and centrifuged under the same conditions to remove the buffer solution. The obtained cell pellet was resuspended in Tris-HCl buffer, PMSF was added to a final concentration of 1mM, and then disrupted in an ice-water bath using an ultrasonic disruptor. The parameters of the ultrasonograph were set as: power 140W, crushing time 10s, interval time 10s and total working time 30min. After the disruption was completed, the disruption solution was centrifuged at 14000rpm for 15min at 4 ℃ to remove the precipitate, and the supernatant was collected into a new EP tube and stored at-80 ℃. If the obtained thallus is not broken immediately, the thallus needs to be stored in a refrigerator at the temperature of minus 80 ℃.

(2) Separation and purification of fusion protein GLEGB by ATPF

Taking a certain amount of bacterial liquid containing fusion protein, surfactant BS-12 and (NH) ₄ ) ₂ SO ₄ Adding into a 20mL colorimetric cylinder and dissolving with distilled water to the scale mark to make the concentration of the surfactant BS-12 be 1.0%, (NH) ₄ ) ₂ SO ₄ Shaking the solution uniformly to be transferred into a flotation tank as a lower phase, adding PEG2000 with the concentration of 50% as an upper phase, carrying out flotation, controlling the flow rate of the flotation at 40mL/min, carrying out the flotation for 20min, collecting upper phase enzyme liquid after bubbles in the flotation tank completely disappear after the flotation is finished, accurately recording the volume, and measuring the enzyme activity recovery rate Ye _a 104.36%, purifying factor PF _a Up to 4.01. The experiments were all performed at room temperature throughout.

2mL of the supernatant enzyme solution was taken, and 0.264g of ammonium sulfate was added to make the concentration of ammonium sulfate in the enzyme solution 1M, and dissolved. The mixture was then heated in a 37 ℃ water bath for 10min and centrifuged at 12000rpm for 15min to remove the supernatant. The pellet was resuspended in precooled Tris-HCl buffer (50mM, pH 8.0), and the resuspended solution was incubated at 4 ℃ for 40min, centrifuged again at 12000rpm for 15min and the pellet discarded. The supernatant is the final purified target enzyme solution. Determination of enzyme activity recovery Ye _a 103.38%, purifying factor PF _a Up to 16.97.

Example 3:

(1) Inducible expression of fusion protein GLEGB

Recombinant plasmids pET-GLEGB, pET-GLE and pET-GLEH were constructed as in step (1) of example 1. The recombinant plasmid was transformed into E.coli BL21 cells to express the fusion gene.

The induced expression of the recombinant plasmid was carried out in E.coli BL21 cells. First, a monoclonal bacterium of the objective transformant was selected in the presence of kanamycin, inoculated into 3mL of LB liquid medium, and cultured overnight at 37 ℃ and 190 rpm. 2mL of the bacterial liquid is taken and transferred into 200mL of liquid LB culture medium containing the kanamycin according to the proportion of 1 percent for amplification culture until the OD600 value is 0.4. The suspension was allowed to stand on ice for 20min and IPTG was added to bring the final concentration to 0.2mM, and the culture was continued overnight at 190rpm at 25 ℃. Then, the cells were centrifuged at 4500rpm for 20min at 4 ℃ to remove the medium, and the pellet was collected. And washed two to three times with 50mM Tris-HCl buffer solution at pH8.0, and centrifuged under the same conditions to remove the buffer solution. The obtained cell pellet was resuspended in Tris-HCl buffer, PMSF was added to a final concentration of 1mM, and then disrupted in an ice-water bath using an ultrasonic disruptor. The parameters of the ultrasonograph were set as: power 140W, crushing time 10s, interval time 10s and total working time 30min. After the disruption was completed, the disruption solution was centrifuged at 14000rpm for 15min at 4 ℃ to remove the precipitate, and the supernatant was collected into a new EP tube and stored at-80 ℃. If the obtained thallus is not broken immediately, the thallus needs to be stored in a refrigerator at the temperature of minus 80 ℃.

(2) Separation and purification of fusion protein GLEGB by ATPF

Taking a certain amount of bacterial liquid containing fusion protein, surfactant BS-12 and (NH) ₄ ) ₂ SO ₄ Adding into a 20mL colorimetric cylinder and dissolving with distilled water to the scale mark to make the concentration of the surfactant BS-12 be 1.5%, (NH) ₄ ) ₂ SO ₄ Shaking the solution uniformly to be transferred into a flotation tank as a lower phase, adding 40% PEG2000 as an upper phase, carrying out flotation, controlling the flow rate of the flotation at 30mL/min and carrying out the flotation for 30min, collecting upper phase enzyme liquid after bubbles in the flotation tank completely disappear after the flotation is finished, accurately recording the volume, and measuring the enzyme activity recovery rate Ye _a 172.86 percent and purification factor PF _a Up to 9.56. The experiments were all performed at room temperature throughout.

2mL of the supernatant enzyme solution was taken, and 0.132g of ammonium sulfate was added thereto so that the concentration of ammonium sulfate in the enzyme solution was 0.5M, and dissolved. The mixture was then heated in a 25 ℃ water bath for 10min and centrifuged at 12000rpm for 15min to remove the supernatant. The pellet was resuspended in precooled Tris-HCl buffer (50mM, pH 8.0), and the resuspended solution was incubated at 4 ℃ for 30min, centrifuged again at 12000rpm for 15min and the pellet discarded. The supernatant is the final purified target enzyme solution. Determination of enzyme activity recovery yield Ye _a Up to 12492%, purification factor PF _a Up to 24.26.

FIG. 2 is a diagram of the phase change mechanism of ELP. The crude enzyme solution of GLEGB enters the upper phase after passing through ATPF to obtain a mixed solution containing GLEGB and PEG2000, and in order to further purify GLEGB, the mechanism of ELP label in fusion protein is continuously utilized to remove PEG2000 through ITC circulation, thereby obtaining the finally purified GLEGB.

FIG. 3 is an SDS-PAGE gel of ATPF in combination with ITC for GLE, GLEH and GLEGB purification. Wherein, lane 1: crude enzyme solution of GLEGB; lane 2: purified GLEGB; lane M: a protein molecular weight marker; lane 3: GLE crude enzyme solution; lane 4: purified GLE; lane 5: crude enzyme solution of GLEH; lane 6: purified GLEH. As can be seen from the figure, GLEGB was purified in the greatest amount and in the best purification effect.

FIG. 4 shows the case of 6 cycles of PEG2000 in GLEGB purified by ATPF combined with ITC technology. The PEG2000 separated in the ITC cycle was used directly in the next flotation system and the procedure was repeated 5 times comparing the differences after several cycles. As can be seen, the 6-time purification effect graphs are substantially consistent. The method shows that the GLEGB fusion enzyme purified by the ATPF-ITC method can not only obtain the target enzyme with higher purity, but also reduce the experiment cost and the environmental pollution, and realize sustainable circulation.

FIG. 5 is a comparison of UV (UV-vis; FIG. 6 a), IR (FT-IR; FIG. 6 b) and CIRCULAR BIOROGRAPHY (CD; FIG. 6 c) spectra of purchased pure Glu and purified GLEGB. It can be seen that there was no significant change in the secondary structure of the GLEGB fusion enzyme compared to β -glucosidase. The addition of ELP and GB labels has no significant influence on the secondary structure of Glu, and ATPF-ITC has no change on enzyme in the process of separating and purifying GLEGB.

Comparative example 1: separation and purification of fusion protein GLEGB by directly using ATPF

Taking a certain amount of bacterial liquid containing fusion protein, surfactant BS-12 and (NH) ₄ ) ₂ SO ₄ Adding into 20mL colorimetric tube, dissolving with distilled water to scale mark, shaking, transferring into flotation cell as lower phase, adding PEG2000 as upper phase, performing flotation, and controlling flotationFlow rate and flotation time. After flotation is finished, collecting upper phase enzyme liquid after bubbles in the flotation tank completely disappear, accurately recording the volume, and measuring the enzyme activity recovery rate Ye _a And purifying factor PF _a 。

A four-factor three-level Box-Behnken (BBD) model is established for the relevant factors (PEG 2000 concentration, surfactant BS-12 concentration, flotation time and flotation flow rate) through response surface design, and an optimal flotation scheme is determined through experiments to obtain a corresponding purification efficiency result. As shown in table 1.

TABLE 1 response surface design experiment results

From the data, it can be seen that when fusion protein GLEGB is directly separated and purified by ATPF, the purification factor PF is _a Generally lower.

Comparative example 2: separation and purification effects of GLEGB directly by using ITC

2mL of the bacterial solution containing the fusion protein was dissolved by adding 0.132g of ammonium sulfate. The mixture was then heated in a water bath at 25 ℃ for 10min and centrifuged at 12000rpm for 15min to remove the supernatant. The pellet was resuspended in precooled Tris-HCl buffer (50 mM, pH 8.0) and the resuspended solution incubated at 4 ℃ for 30min and centrifuged again at 12000rpm for 15min and the pellet discarded. At the moment, the recovery rate Ye of the enzyme activity of the supernatant is _c 78.3 percent, purification factor PF _c Up to 19.1.

By comparison, the combination of the ATPF and ITC methods was much better than the single use of both for purifying the GLEGB fusion enzyme. Research shows that ATPF has obvious effect on improving enzyme activity recovery rate, but the effect is not obvious for purifying factors; and the ITC greatly improves the value of the purification factor, but the recovery rate of the enzyme activity is lower. The combination of the two methods effectively overcomes the defects of the two methods, so that the enzyme activity recovery rate is relatively improved, the purification factor is increased, and the final purification efficiency is supported more strongly.

Comparative example 3: importance of selecting GB labels in ATPF flotation

In the aqueous two-phase flotation (ATPF) process, GLEGB realizes the aim of floating the target enzyme from a lower phase to an upper phase by means of the hydrophobic effect and the bubble adsorption of the GB label, so the hydrophobic effect of the GB label is very important. To demonstrate the hydrophobic effect of the GB tag, the present study compared the flotation effect of several fusion proteins (Glu, GLE and GLG) and the results are shown in table 2. Compared with Glu without a label, the addition of the GB label greatly improves the flotation effect, and successfully verifies the hydrophobic effect of the GB label on the fusion protein.

TABLE 2.4 comparison of the effects of the separation and purification of fusion proteins using ATPF

Fusion proteins	Ye _a	PF _a
			Glu	67.3±0.1354	2.76±0.2692
GLEGB	172.86±0.0384	9.58±0.0142
			GLE	77.54±0.0265	1.87±0.0915
GLG	122.49±0.0329	7.63±0.2343

Comparative example 4: selection of upper phase, lower phase and surfactant in ATPF step

As known from previous studies, glu has an optimum pH of about 5.5 and an optimum reaction temperature of 40-55 ℃. The invention verifies polyethylene glycol (PEG) (600, 2000, 4000, 8000 and 10000) with different molecular weights and different salts (K) ₃ PO4、K ₃ C ₆ H ₅ O ₇ And (NH) ₄ ) ₂ SO ₄ ) And the enzyme activity of Glu under different kinds of surfactants (BS-12 (dodecyl dimethyl betaine), CTAB (cetyl trimethyl ammonium bromide), SDS (sodium dodecyl sulfate) and Triton X-100) changes with time within 2 h. The specific results are shown in FIGS. 6 (a-e).

As can be seen from FIGS. 6 (a-e), the enzyme activity gradually decreased in each PEG with the increase of time. Wherein, PEG 8000 and PEG 10000 has obvious inhibiting effect on enzyme activity compared with PEG with other average molecular weight.

The two-aqueous phase flotation system mainly depends on high-concentration salt, and utilizes the salting-out effect of the salt to maintain two phases to be immiscible, so that the type and concentration of the salt are important factors to be investigated by ATPF. FIG. 7 (a-c) explores Glu in three salts (K) ₃ PO ₄ ，K ₃ C ₆ H ₅ O ₇ And (NH) ₄ ) ₂ SO ₄ ) Stability in (1). Based on that PEG and three kinds of phase separation salt form a proper double-water-phase flotation phase separation system, the concentration of the required salt is different, and according to the verification result, the concentrations of the three kinds of phase separation salt are finally determined to be 30-50 percent (K) ₃ PO ₄ )， 60％-80％(K ₃ C ₆ H ₅ O ₇ ) And 25% -45% ((NH) ₄ ) ₂ SO ₄ ). As can be seen from the figure, withIncreasing time, the activity of Glu in all three salts decreased gradually, but to a different extent in each salt. On the one hand, the optimum pH of Glu is around 5.5, while K ₃ PO ₄ And K ₃ C ₆ H ₅ O ₇ The pH value of the created environment is alkaline, so that the created environment is not beneficial to the survival of Glu; on the other hand, the presence of these two high concentrations of salt requires the anion of the salt to complex more water molecules and thus inhibit enzyme activity. Thus, the present invention selects (NH) ₄ ) ₂ SO ₄ As a phase separated salt of ATPF.

In the process of ATPF floating Glu, the surfactant is added in a certain amount, the main function of the surfactant is to effectively improve the floating efficiency by inhibiting the deformation of bubbles and increasing the stability of the bubbles, and based on the result, the stability of Glu in BS-12, CTAB, SDS and Triton X-100 solutions is verified in the experiment, and the result is shown in FIGS. 8 (a-d). As can be seen from the figure, the anionic and cationic surfactants have obvious influence on the enzyme activity of Glu, while Triton X-100 and BS-12 have no obvious influence on the enzyme activity of Glu within 1h, and especially the BS-12 still retains at least 80% of the enzyme activity after 2 h. Therefore, in general terms, BS-12 is ultimately selected as the surfactant in the separation and purification process of the present embodiment.

Comparative example 5: selection of flotation conditions in ATPF process

FIG. 9 is a graph showing the effect of a series of PEGs of different molecular weights on the separation and purification of GLEGB from ATPF. The results show that as the molecular weight of PEG increases, the flotation efficiency (Ye) _a And PF _a ) Increasing first and then decreasing. Therefore, the present invention selects PEG2000 as the phase forming polymer according to the flotation effect.

After that, the effect of the concentration of PEG2000 on the flotation effect is continuously verified, as shown in fig. 10, the flotation effect also shows a trend of increasing first and then decreasing with the increase of the concentration. Based on this, the present invention selects PEG2000 with a concentration of 30-50%, preferably PEG2000 with a concentration of 40% as the upper phase material for flotation.

The invention verifies that (NH) with different concentrations ₄ ) ₂ SO ₄ (0.25 g/mL-0.45 g/mL) flotation efficiency for purified GLEGB. According to FIG. 11With increasing concentration, ye _a And PF _a Increasing first and then decreasing. The GLEGB flotation to the upper phase mainly depends on salting-out action and hydrophobic adsorption bubbles of GB labels contained in the GLEGB flotation to the upper phase, but the volume of the upper phase is gradually reduced along with the continuous increase of salt concentration, the receptivity to the GLEGB is saturated, and the spatial repulsion of PEG2000 and GLEGB is increased along with the continuous increase of the salt concentration, so that the flotation effect is reduced. Therefore, the final concentration of the phase separation salt in this study was 0.25g/mL to 0.45g/mL ammonium sulfate, preferably 0.35g/mL ammonium sulfate.

The invention continuously verifies the influence of the concentration of BS-12 on the separation and purification of GLEGB by ATPF. According to fig. 12, it is shown that the flotation efficiency is maximized when the concentration of BS-12 reaches 1.5%, and the flotation efficiency is decreased by increasing the concentration of the surfactant. Therefore, the present invention finally selects BS-12 as the surfactant at a concentration of 1.0-2.0%, preferably 1.5%. In order to embody the flotation effect of BS-12, the invention further verifies the effect of not adding the BS-12 flotation fusion protein GLEGB under the same experimental conditions, and the result shows that Ye is added when not adding BS-12 _a And PF _a The values (90.34% + -0.0142 and 4.76 + -0.0430) are much lower than those in the presence of BS-12 (172.86% + -0.0384 and 9.58 + -0.0142), indicating that BS-12 acts as a good foam stabilizer and thus improves the flotation separation.

The gas flow rate and the flotation time are also important factors for investigation in the flotation process, so the invention verifies the effect of separating and purifying GLEGB under different nitrogen flow rates (10-40 mL/min) and flotation times (10-40 min). As can be seen from FIGS. 13 and 14, a nitrogen flow rate of 30mL/min was selected as the optimum flow rate. Moreover, the flotation effect increases with increasing flotation time, reaching the best flotation effect at 30min. Therefore, the final flotation time is 20-40min, wherein 30min is used as the optimal flotation time; the flow rate was chosen to be 20-40mL/min with a nitrogen flow rate of 30mL/min as the optimum flow rate.

Sequence listing

<110> Jiangsu university

<120> method for separating and purifying beta-glucosidase fusion protein

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 481

<212> PRT

<213> Taiwan Coptotermes formosanus

<400> 1

Met Asp Asp Val Asp Asn Asp Thr Leu Val Thr Phe Pro Asp Asp Phe

1 5 10 15

Lys Leu Gly Ala Ala Thr Ala Ser Tyr Gln Ile Glu Gly Gly Trp Asp

20 25 30

Ala Asp Gly Lys Gly Pro Asn Ile Trp Asp Thr Leu Thr His Glu Arg

35 40 45

Pro His Leu Val Val Asp Arg Ser Thr Gly Asp Val Ala Asp Asp Ser

50 55 60

Tyr His Leu Tyr Leu Glu Asp Val Arg Leu Leu Lys Asp Met Gly Ala

65 70 75 80

Glu Val Tyr Arg Phe Ser Ile Ser Trp Ala Arg Ile Leu Pro Glu Gly

85 90 95

His Asp Asn Asn Val Asn Glu Ala Gly Ile Glu Tyr Tyr Asn Lys Leu

100 105 110

Ile Asp Ala Leu Leu Arg Asn Gly Ile Glu Pro Met Val Thr Met Tyr

115 120 125

His Trp Asp Leu Pro Gln Lys Leu Gln Asp Leu Gly Gly Trp Pro Asn

130 135 140

Arg Ile Leu Ala Lys Tyr Ala Glu Asn Tyr Ala Arg Val Leu Phe Ser

145 150 155 160

Asn Phe Gly Asp Arg Val Lys Gln Trp Leu Thr Phe Asn Glu Pro Leu

165 170 175

Thr Phe Met Asp Ala Tyr Ala Ser Asp Thr Gly Met Ala Pro Ser Val

180 185 190

Asp Thr Pro Gly Ile Gly Asp Tyr Leu Thr Ala His Thr Val Ile Leu

195 200 205

Ala His Ala Asn Ile Tyr Arg Leu Tyr Glu Arg Glu Phe Arg Glu Glu

210 215 220

Gln Gln Gly Gln Val Gly Ile Ala Leu Asn Ile His Trp Cys Glu Pro

225 230 235 240

Glu Thr Gly Ser Pro Lys Asp Val Glu Ala Cys Glu Arg Tyr Gln Gln

245 250 255

Phe Asn Leu Gly Ile Tyr Ala His Pro Ile Phe Ser Glu Asn Gly Asp

260 265 270

Tyr Pro Ser Val Leu Lys Ala Arg Val Asp Ala Asn Ser Ala Ser Glu

275 280 285

Gly Tyr Thr Thr Ser Arg Leu Pro Lys Phe Thr Pro Glu Glu Val Ala

290 295 300

Phe Val Asn Gly Thr Tyr Asp Phe Leu Gly Leu Asn Phe Tyr Thr Ala

305 310 315 320

Val Val Gly Arg Asp Gly Val Glu Gly Glu Pro Pro Ser Arg Tyr Arg

325 330 335

Asp Met Gly Thr Ile Thr Ser Gln Asp Pro Glu Trp Pro Glu Ser Ala

340 345 350

Ser Ser Trp Leu Arg Val Val Pro Trp Gly Phe Arg Lys Glu Leu Asn

355 360 365

Trp Ile Ala Asn Glu Tyr Gly Asn Pro Pro Ile Phe Ile Thr Glu Asn

370 375 380

Gly Phe Ser Asp Tyr Gly Gly Val Asn Asp Thr Asn Arg Val Leu Tyr

385 390 395 400

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

405 410 415

Val Asn Val Ile Gly Tyr Thr Ala Trp Ser Leu Ile Asp Asn Phe Glu

420 425 430

Trp Leu Arg Gly Tyr Thr Glu Arg Phe Gly Ile His Ala Val Asn Phe

435 440 445

Ile Asp Pro Ser Arg Pro Arg Thr Pro Lys Glu Ser Ala Arg Val Leu

450 455 460

Thr Glu Ile Phe Lys Thr Arg Gln Ile Pro Glu Arg Phe Arg Asp Ala

465 470 475 480

Ser

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

1 5 10 15

Leu

<210> 3

<211> 252

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1 5 10 15

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

20 25 30

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

35 40 45

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

50 55 60

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

65 70 75 80

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

85 90 95

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

100 105 110

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

115 120 125

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

130 135 140

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

145 150 155 160

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

165 170 175

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

180 185 190

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

195 200 205

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

210 215 220

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

225 230 235 240

Val Pro Gly Val Gly Val Pro Gly Val Gly Lys Leu

245 250

<210> 4

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

His Asn Trp Tyr His Trp Trp Pro His

1 5

Claims

1. A method for separating and purifying a fusion protein GLEGB (Glu-linker-ELP-GB), which is characterized by comprising the following steps:

(1) Induced expression of fusion protein GLEGB: connecting the nucleotide sequence of ELP and GB-like polypeptides to the nucleotide sequence of Glu-beta-glucosidase by using the nucleotide sequence of linker-encoding connecting peptide to construct a recombinant plasmid pET-GLEGB, and transforming the recombinant plasmid into host cells to express fusion protein;

the amino acid sequence of the linker is shown in SEQ.ID NO. 2;

the amino acid sequence of the elastin-like protein ELP is shown in SEQ.ID.NO. 3;

the GB is graphene binding peptide, and the amino acid sequence of the GB is shown in SEQ.ID.NO.4;

(2) Separating and purifying fusion protein GLEGB by aqueous two-phase flotation (ATPF): mixing bacterial liquid containing fusion protein, surfactant BS-12 and (NH) ₄ ) ₂ SO ₄ Mixing to serve as a lower phase, taking an upper phase as PEG2000, performing flotation, respectively collecting upper and lower phase solutions after flotation, recording the volume, and measuring the enzyme activity and the protein content in the upper phase and the lower phase after flotation; in the lower phase (NH) ₄ ) ₂ SO ₄ The concentration of (b) is 0.25g/mL-0.45g/mL; the concentration of the surfactant is 1.0-2.0%; the volume ratio of the lower phase to the upper phase is 5:1; wherein the using amount of the bacterial liquid containing the fusion protein in the lower phase accounts for 1/5 of the total volume of the lower phase; the concentration of PEG2000 in the upper phase is 30-50%; the flotation condition is that nitrogen is introduced to carry out flotation for 20-40min under the condition that the flow rate is 20-40 mL/min;

(3) Separation and purification of fusion protein GLEGB by combining ATPF with reverse switching cycle (ITC): adding ammonium sulfate into the collected upper phase mixed solution, heating in water bath, and centrifuging to remove supernatant; then, the sediment is resuspended by using Tris-HCl buffer solution, and is incubated for a certain time at low temperature, and the impurities are removed by centrifugation, so that the purified target enzyme solution is obtained.

2. The method according to claim 1, wherein in step (3), the ammonium sulfate is added to the upper phase enzyme solution so that the final concentration of ammonium sulfate is 0.1 to 1M.

3. The method according to claim 1, wherein in step (3), the water bath is heated for 10min at a temperature of 15-37 ℃.

4. The method according to claim 1, wherein in step (3), the resuspended solution is incubated under conditions such that: 4 ℃ for 10-40min.