Preparation process of acinetobacter baumannii extracellular protein OmpA

Abstract

The invention discloses a preparation process of acinetobacter baumannii extracellular protein OmpA, which comprises the following steps: (1) seeds are inoculated into a seed culture medium for primary culture, then secondary culture is carried out, IPTG (isopropyl-beta-d-thiogalactoside) is added, induction is carried out for 3-5 hours, and the culture medium comprises yeast powder, a nitrogen source and salt; (2) centrifuging, collecting thalli, and crushing the thalli; (3) dissolving and solubilizing the inclusion body, namely dissolving 1.0 g of precipitate in 10mL of solution, centrifuging, and collecting supernate; (4) anion amp; carrying out cation chromatography; (5) dilution and renaturation: diluting with a renaturation solution with the pH value of 7.5 by using 20mM PB, renaturating overnight, centrifuging, and waiting for column loading; (6) cation chromatography; and (7) dialyzing and changing the solution. The protein purity (SDS-PAGE) of the acinetobacter baumannii extracellular protein OmpA prepared by the method disclosed by the invention is more than 99%, and the protein yield is 15.4 mg / g inclusion body.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a preparation process of Acinetobacter baumannii extramembrane protein OmpA. Background Art

[0002] Among the Omps of Acinetobacter baumannii, OmpA is the most intensively studied virulence factor. OmpA plays a key role in regulating the adhesion, invasiveness and biofilm formation of Acinetobacter baumannii and the host immune response. Excessive production of OmpA is an independent risk factor for mortality from nosocomial pneumonia and bacteremia caused by Acinetobacter baumannii. In addition, the OmpA expression level measured by qRT-PCR can be used as a rapid diagnostic indicator for antibiotic-resistant Acinetobacter baumannii, and the results are highly consistent with the traditional MIC analysis results.

[0003] With the resolution of the native structure of OmpA, researchers found that the amino acid sequences of Acinetobacter baumannii OmpA (AbOmpA) from multiple clinical isolates were highly conserved (>89%), while they were not homologous to the human proteome. By comparing various OmpA-like proteins, two conserved amino acids, Arg286 and Asp271, in the C-terminal domain of OmpA were identified, which non-covalently bind to diaminopimelic acid (DAP), a component of peptidoglycan (PNG). This interaction suggests that OmpA plays a key role in maintaining bacterial integrity. In general, in GNB species, OmpA stabilizes its structure by self-dimerization, thereby preventing the cleavage of the curved connection between the β-barrel structure and the periplasm.

[0004] Acinetobacter baumannii OmpA has the following advantages: it is essential for Acinetobacter baumannii to adhere to and invade epithelial cells; the penetration of small molecule antibiotics through Acinetobacter baumannii OmpA is of great significance for bacterial resistance; Acinetobacter baumannii OmpA has strong immunogenicity and immune protection as a vaccine antigen; Acinetobacter baumannii OmpA may have anti-infection and anti-cancer activity by regulating the activity of immune cells.

[0005] The prior art lacks a preparation process for high-purity Acinetobacter baumannii extramembrane protein OmpA. Therefore, providing a preparation process for high-purity Acinetobacter baumannii extramembrane protein OmpA is particularly important for the application of Acinetobacter baumannii extramembrane protein OmpA in various aspects. Summary of the invention

[0006] The object of the present invention is to provide a preparation process of Acinetobacter baumannii extramembrane protein OmpA, so as to solve the technical problem of lack of preparation process of high-purity Acinetobacter baumannii extramembrane protein OmpA in the prior art.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The preparation process of the Acinetobacter baumannii extramembrane protein OmpA provided by the present invention comprises the following steps:

[0009] (1) inoculating the seeds into a seed culture medium for primary culture, and then inoculating the seeds into a culture medium for secondary culture at a ratio of 1:60, and when the OD600 reaches 0.6-0.9, adding IPTG at a ratio of 1:1000 for induction for 3-5 hours, wherein the culture medium includes yeast powder, a nitrogen source and salt;

[0010] (2) collecting the cells after centrifugation and breaking the cells;

[0011] (3) Inclusion body dissolution and solubilization: 1.0 g of precipitate was added to 10 mL of solution for dissolution, and the supernatant was collected after centrifugation;

[0012] (4) Anion & cation chromatography;

[0013] (5) Dilution and renaturation: dilute with 20 mM PB, pH 7.5 renaturation solution and renature overnight, centrifuge and load onto the column;

[0014] (6) Cationic chromatography;

[0015] (7) Dialysis and fluid exchange: Based on the target protein volume enriched by the chromatography column (6), a dialysis bag with a molecular weight cutoff of 14KD was selected, the dialysis multiple was 1:100, and the dialysis was performed twice at 4-8°C, with fluid exchange in the middle. The single dialysis time was more than 5 hours, and finally the original solution of the extramembrane protein OmpA of Acinetobacter baumannii was obtained.

[0016] Furthermore, the preceding step (1) further includes: plasmid construction, protein expression: synthesizing the OmpA expression sequence, using the gene sequence as a template, PCR amplifying the target gene sequence, and constructing it into a pET30a expression vector with Xho I and Nde I restriction enzymes, transforming the constructed OmpA into the E. coli expression strain BL21 (DE3) and inducing expression to obtain seed bacteria.

[0017] Furthermore, the amino acid sequence of OmpA is shown in SEQ ID NO:1, and the nucleotide sequence of OmpA is shown in SEQ ID NO:2.

[0018] Furthermore, the culture medium formula in (1) is: 5 g / L yeast powder, 10 g / L peptone, and 10 g / L sodium chloride; the seed culture medium also contains 50 mg / ml kanamycin.

[0019] Furthermore, in the step (2), the bacterial cells are disrupted by diluting and dissolving the bacterial cells with 20 mM Tris, pH=8.0, and then homogenizing, centrifuging, and collecting the precipitate.

[0020] Furthermore, the centrifugation conditions in (2) are: centrifugal force 8000g, 10min, and centrifugal temperature 2-8°C.

[0021] Furthermore, the (2) bacterial cell disruption is as follows: dilute and dissolve the bacterial cells and 20mM pH=8.0 Tris at a ratio of 1:10, stir and dissolve completely, average 800±50Bar for 3 times, centrifuge at 15000g, 2-8°C for 15min, and collect the precipitate; add 20mM Tris, pH 8.0 at a ratio of 1:10 to dilute and dissolve the collected precipitate, centrifuge at 15000g, 4°C, 15min, and collect the precipitate. Repeat the above operation once. Put the washed inclusion bodies into -20°C for use.

[0022] Furthermore, the solution in (3) is 20 mM PB+8 M urea at pH 7.50.

[0023] Furthermore, the (3) is specifically as follows: dissolving the precipitate in a ratio of 1.0 g to 10 mL of solution, crushing the precipitate using an ultrasonic crusher, 400 W for 10 min, working for 6 s, off for 4 s, stirring at 4° C. and 150 rpm for 2 h, centrifuging at 15,000 g and 2-8° C. for 30 min, and collecting the supernatant.

[0024] Furthermore, the specific embodiment of (4) is:

[0025] Pretreatment: 1.5 ml / min, 0.5 M NaOH for 2-3 CV, then 20 mM PB, 8 M urea, 1000 mM NaCl, pH 7.50 for 2-5 column volumes;

[0026] Equilibration: 2 ml / min, wash 2-3 column volumes with 20 mM PB, 8 M urea, pH 7.50;

[0027] Sample loading: 2 ml / min injection, collect flow-through;

[0028] Elution: 2 ml / min, 20 mM PB, 8 M urea, 1000 mM NaCl, pH 7.50, wash 2-3 column volumes;

[0029] Post-treatment: 2 ml / min, 0.4 M acetic acid + 1000 mM NaCl → 20 mM Tris → 0.5 M NaOH → 2 M NaCl → purified water → 20% ethanol, wash for at least 2 column volumes in sequence.

[0030] 7. The process for preparing the extramembrane protein OmpA of Acinetobacter baumannii according to claim 1, characterized in that the step (6) is as follows:

[0031] Pretreatment: 3 ml / min, 0.5 M NaOH → 20 mM PB, 1000 mM NaCl, pH 7.50, repeat for at least 2-3 column volumes;

[0032] Pre-equilibrium: 5 ml / min, 20 mM PB, pH 7.50, wash 5 column volumes;

[0033] Sample loading: 5ml / min injection;

[0034] Post-equilibrium: 5 ml / min, 20 mM PB, pH 7.50, wash 5 column volumes;

[0035] Wash: 5 ml / min, 20 mM PB, 500 mM NaCl, pH 7.50, 5 column volumes;

[0036] Elution: 5 ml / min, 20 mM PB, 1000 mM NaCl, pH 7.50, wash for 5 column volumes, collect the eluate;

[0037] Post-treatment: 2 ml / min, 0.5 M NaOH → 2 M NaCl → water → 20% ethanol, wash for at least 2 column volumes in sequence.

[0038] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:

[0039] The invention provides a preparation process for the Acinetobacter baumannii extramembrane protein OmpA. The prepared Acinetobacter baumannii extramembrane protein OmpA has a protein purity (SDS-PAGE) of more than 99% and a protein yield of 15.4 mg / g inclusion body. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0041] Figure 1 This is a diagram of the electrophoresis result of Y-ABF-OmpA in an embodiment of the present invention;

[0042] Figure 2 It is the electrophoresis result diagram of Y-ABP-OmpA of the embodiment of the present invention. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] Example 1

[0049] Plasmid construction and protein expression: The OmpA expression sequence was synthesized, and the target gene sequence was amplified by PCR using the gene sequence as a template. The sequence was cut with Xho I and Nde I to construct the pET30a expression vector. The constructed OmpA was transformed into the E. coli expression strain BL21 (DE3) and induced for expression to obtain seed bacteria.

[0050] The amino acid sequence of OmpA is shown in SEQ ID NO:1, and the nucleotide sequence of OmpA is shown in SEQ ID NO:2.

[0051] SEQ ID NO: 1:

[0052] MANAGVTVTPLLLGYTFQDSQHNNGGKDGNLTNGPELQDDLFVGAALGIELTPWLG

[0053] FEAEYNQ

[0054] VKGDVDGASAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYDFDGV

[0055] NRGTRGTS

[0056] EEGTLGNAGVGAFWRLNDALSLRTEARATYNADEEFWNYTALAGLNVVLGGHLKP

[0057] AAPVEVA

[0058] PVEPTPVTPQPQELTEDLNMELRVFFDTNKSNIKDQYKPEIAKVAEKLSEYPNATARI

[0059] EGHTDNT

[0060] GPRKLNERLSLARANSVKSALVNEYNVDASRLSTQGFAWDQPIADNKTKEGRAMNR

[0061] RVFATITG

[0062] SRTVVVQPGQEAAAPAAAQ

[0063] SEQ ID NO:2

[0064] ATGGCTAATGCTGGCGTAACAGTTACTCCATTATTGCTTGGTTACACTTTCCAAGA

[0065] CAGCCAAC

[0066] ACAACAATGGCGGTAAAGATGGTAACTTAACTAACGGTCCTGAGTTACAAGACG

[0067] ATTTATTCG

[0068] TTGGCGCAGCTCTTGGTATCGAGTTAACTCCATGGTTAGGTTTCGAAGCTGAATA

[0069] INVITED

[0070] TAAAGGCGACGTAGACGGCGCTTCTGCTGGTGCTGAATATAAACAAAACAAAT

[0071] CAACGGTA

[0072] ACTTCTATGTTACTTCTGATTTAATTACTAAAAACTACGACAGCAAAATCAAGCC

[0073] GTACGTATT

[0074] ATTAGGTGCTGGTCACTATAAATACGACTTTGATGGCGTAAACCGTGGTACACGT

[0075] GGTACTTCT

[0076] GAAGAAGGTACTTTAGGTAACGCTGGTGTTGGTGCTTTTCTGGCGCTTAAACGACG

[0077] CTTTATCTC

[0078] TTCGTACTGAAGCTCGTGCTACTTATAATGCTGATGAAGAGTTCTGGAACTATAC

[0079] AGCTCTTGC

[0080] TGGCTTAAACGTAGTTCTTGGTGGTCACTTGAAGCCTGCTGCTCCTGTAGTAGAA

[0081] GTTGCTCCA

[0082] GTTGAACCAACTCCAGTTACTCCACAACCACAAGAGTTAACTGAAGACCTTAAC

[0083] TGGAACTT

[0084] CGTGTGTTCTTTGTAGACTAACAAATCAAACATCAAAGACCAATACAAGCCAGAAA

[0085] TTGCTAAA

[0086] GTTGCTGAAAAAATTATCTGAATACCCTAACGCTACTGCACGTATCGAAGGTCACA

[0087] CAGATAAC

[0088] ACTGGTCCACGTAAGTTGAACGAACGTTTATCTTTAGCTCGTGCTAACTCTGTTAA

[0089] ATCAGCCTCT

[0090] TGTAAACGAATACAACGTTGATGCTTCTCGTTTGTCTACTCAAGGTTTCGCTTGGG

[0091] ATCAACCG

[0092] ATTGCTGACAACAAAACTAAAGAAGGTCGTGCTATGAACCGTCGTGTATTCGCGA

[0093] CAATCACT

[0094] GGTAGCCGTACTGTAGTAGTTCAACCTGGTCAAGAAGCGGCAGCTCCTGCAGCAGCTCAATAA

[0095] (1) Shake flask inoculation culture

[0096] Culture medium formula: yeast powder (5g / L), peptone (10g / L), sodium chloride (10g / L), sterilization temperature 121℃, 20min.

[0097] Use 250ml / bottle of sterilized culture medium. Use a pipette to inoculate the seeds into the seed culture medium containing 50mg / ml kanamycin, a total of 1 bottle. Place the inoculated culture bottle in a shaker. Set the culture temperature to 37℃, the speed to 220rpm, and culture for 16 hours. Take out the culture bottle in the shaker and inoculate it into the culture medium for the second time, inoculating at a ratio of 1 to 60. When OD600 reaches 0.6-0.9, add 1M IPTG at a ratio of 1 to 1000 and induce for 3-5 hours.

[0098] (2) Collect and crush the cells after centrifugation

[0099] Use a centrifuge to centrifuge at a centrifugal force of 8000g for 10 min and a centrifugal temperature of 2-8°C, discard the supernatant, and collect the bacteria.

[0100] Take 20g of bacteria, add 20mm Tris (pH=8.0) at a ratio of 1:10 to dilute and dissolve, stir to dissolve completely, average 800±50Bar 3 times, centrifuge at 15000g, 2-8℃ for 15min, and collect the precipitate. Add 20mMTris, pH 8.0 at a ratio of 1:10 to dilute and dissolve the collected precipitate, centrifuge at 15000g, 4℃, 15min, and collect the precipitate. Repeat the above operation once. Put the washed inclusion bodies into -20℃ for use.

[0101] (3) Inclusion body dissolution and solubilization

[0102] Take out the reserved inclusion bodies from -20℃, and dissolve them in the ratio of 1.0g precipitate to 10mL solution (20mM PB+8M Urea, pH7.50). Take 10.4g inclusion bodies this time. Use ultrasonic crusher to crush, 400W, 10min. Work for 6s, off for 4s. Stir at 4℃, 150rpm for 2h, centrifuge at 15000g, 2-8℃ for 30min. Collect the supernatant.

[0103] (4) Anion & Cation Chromatography (Q-Mustang & SP-FF) XK16 / 20

[0104] Pretreatment: 1.5 ml / min, 0.5 M NaOH to flush Q-Mustang & SP-FF for 2-3 CV, then flush with 20 mM PB, 8 M Urea, 1000 mM NaCl, pH 7.50 for 2-5 column volumes.

[0105] Equilibration: 2 ml / min, flush with 20 mM PB, 8 M Urea, pH 7.50 for 2-3 column volumes.

[0106] Sample loading: 2 ml / min injection, collect the flow-through.

[0107] Elution: 2 ml / min, 20 mM PB, 8 M Urea, 1000 mM NaCl, pH 7.50 for 2-3 column volumes.

[0108] Post-treatment: 2 ml / min, 0.4 M Hac + 1000 mM NaCl → 20 mM Tirs → 0.5 M NaOH → 2 M NaCl → purified water → 20% ethanol, wash in sequence for at least 2 column volumes.

[0109] (5) Dilution and renaturation

[0110] The refolding solution was 20 mM Pb, pH 7.5, taken out from 4°C, stirred in an ice-water bath at 100 rpm, diluted 10 times, and refolded overnight for 16 hours. The refolding solution was taken out, centrifuged at 5000 g, 4°C for 10 minutes, and loaded onto the column.

[0111] (6) Cationic chromatography (CM-FF) XK16 / 20

[0112] Pretreatment: 3 ml / min, 0.5 M NaOH → 20 mM PB, 1000 mM NaCl, pH 7.50, repeat in sequence for at least 2-3 column volumes.

[0113] Pre-equilibration: 5 ml / min, 20 mM PB, pH 7.50, wash for 5 column volumes.

[0114] Sample loading: 5ml / min injection.

[0115] Post-equilibrium: 5 ml / min, 20 mM PB, pH 7.50, wash for 5 column volumes.

[0116] Wash: 5 ml / min, 20 mM PB, 500 mM NaCl, pH 7.50, 5 column volumes

[0117] Elution: 5 ml / min, 20 mM PB, 1000 mM NaCl, pH 7.50, wash for 5 column volumes, and collect the eluate.

[0118] Post-treatment: 2 ml / min, 0.5 M NaOH → 2 M NaCl → water → 20% ethanol, wash for at least 2 column volumes in sequence.

[0119] (7) Dialysis fluid exchange

[0120] According to the target protein volume enriched by the chromatography column (6), a dialysis bag with a molecular weight cutoff of 14KD was selected, the dialysis multiple was 1:100, and the dialysis was performed twice at 4-8°C, with the liquid changed in the middle. The single dialysis time was more than 5 hours, and finally the original solution of the extramembrane protein OmpA of Acinetobacter baumannii was obtained.

[0121] like Figure 1-2 As shown, the protein purity (SDS-PAGE) of the obtained Acinetobacter baumannii extracellular protein OmpA stock solution was above 99%, and the protein yield was 15.4 mg / g inclusion body.

[0122] Finally, it should be noted that:

[0123] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for preparing the extramembrane protein OmpA of Acinetobacter baumannii, characterized in that: The following steps are involved: (1) Inoculating the seeds into a seed culture medium for primary culture, and then inoculating them into a culture medium at a ratio of 1:60 for secondary culture. When the OD600 reaches 0.6-0.9, adding IPTG at a ratio of 1:1000 for induction for 3-5 hours. The culture medium includes yeast powder, nitrogen source and salt; (2) Collect and disrupt the cells after centrifugation; (3) Inclusion body dissolution and solubilization: 1.0 g of precipitate was added to 10 mL of solution for dissolution, and the supernatant was collected after centrifugation; (4) Anion & cation chromatography; (5) Dilution and renaturation: dilute with 20 mM PB, pH 7.5 renaturation solution and renature overnight, centrifuge and load onto the column; (6) Cationic chromatography; (7) Dialysis and fluid exchange: Based on the target protein volume enriched by the chromatography column (6), a dialysis bag with a molecular weight cutoff of 14KD was selected, the dialysis multiple was 1:100, and the dialysis was performed twice at 4-8°C, with fluid exchange in the middle. The single dialysis time was more than 5 hours, and the final dialysis solution of the extramembrane protein OmpA of Acinetobacter baumannii was obtained.

2. The preparation process of the Acinetobacter baumannii extramembrane protein OmpA according to claim 1, characterized in that: The aforementioned (1) subsequent step also includes: plasmid construction, protein expression: synthesizing the OmpA expression sequence, using the gene sequence as a template, PCR amplifying the target gene sequence, and constructing it into the pET30a expression vector with Xho I and Nde I enzymes, transforming the constructed OmpA into the E. coli expression strain BL21 (DE3) and inducing expression to obtain seed bacteria.

3. The preparation process of the Acinetobacter baumannii extramembrane protein OmpA according to claim 1, characterized in that: The culture medium formula of (1) is: 5 g / L yeast powder, 10 g / L peptone, and 10 g / L sodium chloride; the seed culture medium also contains 50 mg / ml kanamycin.

4. The preparation process of the Acinetobacter baumannii extramembrane protein OmpA according to claim 1, characterized in that: In the above (2), the bacterial cells are disrupted by diluting and dissolving the bacterial cells with 20 mM Tris, pH=8.0, and then homogenizing, centrifuging, and collecting the precipitate.

5. The preparation process of the Acinetobacter baumannii extramembrane protein OmpA according to claim 1, characterized in that: The solution in (3) is 20 mM PB + 8 M urea at pH 7.

50.

6. The preparation process of the Acinetobacter baumannii extramembrane protein OmpA according to claim 1, characterized in that: Specifically, (4) is: Pretreatment: 1.5 ml / min, 0.5 M NaOH for 2-3 CV, then 20 mM PB, 8 M urea, 1000 mM NaCl, pH 7.50 for 2-5 column volumes; Equilibration: 2 ml / min, wash with 20 mM PB, 8 M urea, pH 7.50 for 2-3 column volumes; Sample loading: 2 ml / min injection, collect flow-through; Elution: 2 ml / min, 20 mM PB, 8 M urea, 1000 mM NaCl, pH 7.50, wash 2-3 column volumes; Post-treatment: 2 ml / min, 0.4 M acetic acid + 1000 mM NaCl → 20 mM Tirs → 0.5 M NaOH → 2 M NaCl → purified water → 20% ethanol, wash for at least 2 column volumes in sequence.

7. The process for preparing the Acinetobacter baumannii extramembrane protein OmpA according to claim 1, characterized in that: Specifically, (6) is: Pretreatment: 3 ml / min, 0.5 M NaOH → 20 mM PB, 1000 mM NaCl, pH 7.50, repeat for at least 2-3 column volumes; Pre-equilibrium: 5 ml / min, 20 mM PB, pH 7.50, wash 5 column volumes; Sample loading: 5ml / min injection; Post-equilibrium: 5 ml / min, 20 mM PB, pH 7.50, wash 5 column volumes; Wash: 5 ml / min, 20 mM PB, 500 mM NaCl, pH 7.50, 5 column volumes; Elution: 5 ml / min, 20 mM PB, 1000 mM NaCl, pH 7.50, wash for 5 column volumes, collect the eluate; Post-treatment: 2 ml / min, 0.5 M NaOH → 2 M NaCl → water → 20% ethanol, wash for at least 2 column volumes in sequence.

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