CN117466983A

CN117466983A - Mutant protein S27L of serine mutation at 27 th site of streptavidin and application thereof

Info

Publication number: CN117466983A
Application number: CN202311231172.5A
Authority: CN
Inventors: 李洪涛; 邬文峰; 金瑞; 何红梅; 杨婧
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-02-14
Filing date: 2022-02-14
Publication date: 2024-01-30
Also published as: CN114478726A; CN114478726B; CN117659145B; CN117264035A; CN117264035B; CN117659145A

Abstract

The invention discloses a mutant protein S27L with serine mutation at the 27 th site of streptavidin and application thereof, wherein the mutant protein is wild type streptavidin, amino acids at the 44-47 th site of the streptavidin are VTAR, and serine mutation at the 27 th site of the wild type streptavidin is different amino acids, so that the binding force between the mutated protein and Strep tag II is increased, the purification capability of the mutant protein on single Strep and Twain Strep fusion proteins is improved, and meanwhile, the binding with Biotin is weakened, so that the effect of the Biotin is reversible, and the Biotin can be reused after elution, thereby being better used for purifying the Biotin modified protein and Strep tag II tag protein.

Description

Mutant protein S27L of serine mutation at 27 th site of streptavidin and application thereof

Technical Field

The invention relates to mutant proteins with streptavidin mutations, which can bind biotin reversibly after mutation, and thus can be used for protein purification, regeneration and reuse.

Background

Streptavidin (i.e., strepavidin) has a strong non-covalent binding force to Biotin (Biotin), and the dissociation equilibrium constant K of wild-type Streptavidin binding Biotin _d At 10 ^-14 mol/L is the strongest non-covalent interaction known in nature at present, so that the molecular biological method has wide application in the field of molecular biology, and the application fields comprise: affinity chromatography, living cell fluorescence imaging, proteomics, immobilization of biotinylase, and the like. Although the field of application of streptavidin is wide, the requirement for more refined properties of streptavidin, such as lower affinity and higher dissociation constant for affinity chromatography, is specific to each application, so that the target molecule can be effectively eluted from the microsphere containing streptavidin. Since wild-type streptavidin binds very strongly to Biotin (Biotin) modified proteins, it is necessary to heat at 95 ℃ in a buffer solution containing high concentration of Biotin (Biotin) under very severe conditions to elute part of the proteins, and thus it cannot be used for non-denaturing affinity chromatography of Biotin (Biotin) modified proteins; the affinity purification application of wild streptavidin is to use the strong affinity of the wild streptavidin with peptide segment SBP (amino acid sequence MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) with length of 38 amino acids to dissociate equilibrium constant K _d Is 10 ^-9 mol/L, SBP peptide fragment is used as affinity purification tag fusionThe N-segment or C-end of the target protein is subjected to competitive elution by using Biotin, but the disadvantage of the application is that the Biotin and the wild-type streptavidin have ultra-strong binding force, so that the wild-type streptavidin cannot be effectively regenerated, and the application of the SBP label is limited. In order to expand the range of applications for streptavidin, it is necessary to modify streptavidin to weaken its binding force with Biotin.

Voss et al mutation of amino acids 44-47 of streptavidin to give Streptactin mutant (amino acids 44-47 have the sequence Val-Thr-Ala-Arg, truncated sequence of wild-type streptavidin at positions 15-139) which specifically binds to the short peptide Strep tag II (peptide fragment sequence: trp-Ser-His-Pro-Gln-Phe-Glu-Lys) which binds to dissociation equilibrium constant K of Strep tag II _d At 10 ^-7 mol/L, but its dissociation equilibrium constant for biotin, K _d Still at 10 ^-11 ～10 ^-12 mol/L such that its binding to Biotin is still irreversible; wong et al mutated serine (Ser) at position 27 of wild-type streptavidin to alanine (Ala) (S27A), glycine (Gly) at position 48 to threonine (Thr) (G48T) to give SAVSBPM18 mutant with dissociation equilibrium constant K for Biotin _d Rise 10 ^-8 mol/L, so that the binding of Biotin to SAVSBPM18 becomes reversible.

Because SBP tags are longer in length, the application is limited, the most widely used Strep tag and Strep tag at present, but the combination of single Strep tag II and Strep tag is still not strong enough, so that two Strep tags II are connected in series in the actual use process (namely Twainstep tag, peptide fragment sequence: trp-Ser-His-Pro-Gln-Phe-Glu-Lys- (Gly-Gly-Gly-Ser) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys encoding a nucleotide as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO. 2) such that the dissociation equilibrium constant K is _d Up to 10 ^-9 mol/L, and greatly enhances the binding force. However, strepTactin still has the following disadvantages when used for purifying streptag II tagged fusion proteins, particularly, the StrepTactin is difficult to regenerate after eluting with biotin, and the StrepTactin is regenerated by using denaturants, strong acids, strong bases and the like, so that the service life of the StrepTactin is reduced; the elution of the target protein is usually carried out byExpensive desulphated biotin is used to replace biotin for eluting the target protein (Schmidt et al, 2007), but because the price of desulphated biotin is far higher than that of biotin and the use concentration is higher, the use cost is increased, and the method is unfavorable for industrial production.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a mutant protein having a mutation of serine at position 27 and having VATR at amino acids 44 to 47 of streptavidin wild-type streptavidin; it is another object of the present invention to provide an immobilized complex of a mutein comprising a serine mutation at position 27 of said streptavidin; the third object of the invention is to provide the use of the mutant protein of the serine mutation at position 27 of streptavidin or the immobilized complex in purification of Strep tag II or Twainstrap tag proteins; the fourth object of the present invention is to provide the use of said streptavidin mutein or said immobilized complex for the purification of biotin-modified proteins; the fifth object of the present invention is to provide a method for purifying Strep tag II or Twittrep tag protein using the mutant protein of serine at position 27 of streptavidin.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. a mutant protein S27L of the 27 th serine mutation of the streptavidin, wherein the mutant protein is formed by taking the amino acid residues from 44 th to 47 th amino acid residues of the wild-type streptavidin as VTAR, and the serine mutation at the 27 th serine of the wild-type streptavidin is formed by taking leucine as S27L; the amino acid sequence is shown as SEQ ID NO. 24.

2. And a fixed complex of the mutant protein of the 27 th serine mutation of the streptavidin and the microsphere.

3. The application of the mutant protein of the 27 th serine mutation of the streptavidin or the immobilized complex in purifying Strep tag II or Twittrep tag protein.

4. The mutant protein of the 27 th serine mutation of the streptavidin or the crosslinked complex is applied to purifying biotin modified protein.

5. The method for purifying Strep tag II or Twittrep tag protein by utilizing the mutant protein of the serine mutation at the 27 th position of streptavidin comprises the steps of combining a lysate containing Strep tag II or Twittrep tag protein with a fixed complex of the mutant protein, flushing with a buffer solution, eluting with 5-10mM Biotin buffer solution, and collecting an eluent.

The preferred method for purifying Strep tag II or Twittrep tag protein by using the mutant protein of the serine mutation at the 27 th position of streptavidin is characterized by comprising the following steps of: the Biotin buffer solution also comprises the following steps of recycling after elution: and adding buffer solution into the immobilized complex purified by Strep tag II or Twittrep tag protein for washing, and then reusing.

The invention has the beneficial effects that: the invention provides a mutant protein of the 27 th serine mutation of streptavidin, which is characterized in that the serine (Ser) of the 27 th serine of the wild type streptavidin is mutated into mutants with different amino acids, the binding force between the mutated protein and Strep tag II is increased, the purification capability of Strep and Twain Strep fusion proteins is improved, the binding force between the mutated protein and Biotin is weakened, the Biotin and the Biotin become reversible, and the Biotin can be washed and regenerated for reuse after elution, so that the mutant protein can be better used for purifying Biotin modified proteins and Strep tag II, biotin modified proteins can be eluted by Biotin, and Strep tag mut can be reused after washing and regeneration by buffer solution after elution.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 shows the selection of the mutant enriched in Twittrep modified proteins at position 27 (A: single comparison of the effects of Stepactin beams and S27T, S27A, S27G, S27E, S27Q, S27W, S27C, S27H beams to purify Twittrep-eGFP; B: single comparison of the effects of Stepactin beams and S27T, S27N, S27V, S27M, S27P, S27L, S27K, beams to purify Twittrep-eGFP; C: single comparison of the effects of Stepactin beams and S27T, S27Y, S27I, S27D, S27PR, S27F beams to purify Twittrep-eGFP).

FIG. 2 is a graph showing the comparison of the effect of the enrichment of the 27-bit mutant with Twittrep modified protein on the regeneration of 3 times (A: S27T reads and S27A, S27G reads purified Twittrep-eGFP (S27T 1-3: three experiments on the regeneration of S27T reads purified Twittrep-eGFP; S27A 1-3S 27ABeads purified Twoinstrep-eGFP was regenerated three times; S27G 1-3S 27G Beads purified Twittrep-eGFP regeneration three experiments: B.S 27T-Beads and S27H, S27Q-Beads purification Twist-eGFP (S27T 1-3: three experiments for Twist-eGFP purification, S27H 1-3: three experiments for Twist-eGFP purification, S27Q 1-3: three experiments for GFP purification, S27Q-Beads purification, twist-eGFP regeneration), C.S 27T-Beads and S27L, twist-Beads purification, twist-eGFP effect comparison graph (S27T 1-3: twist-Beads purification, S27L 1-3: three experiments for Twist-eGFP regeneration, S27L 1-3: three experiments for GFP purification, S27P 1-3: three experiments for GFP purification, S27T-Beads purification, twist-27P-eGFP regeneration, and Twist-27T 1-3: three experiments for Twist-sBeads purification, twist-27F-27T 1-27T-3: three experiments for Twist-GFP purification, comparison of the effect of S27K Beads purified Twittrep-eGFP (three experiments for regeneration of S27T1-3: S27T Beads purified Twittrep-eGFP; three experiments for regeneration of S27N 1-3: S27N Beads purified Twittrep-eGFP; three experiments for regeneration of S27K 1-3: S27K Beads purified Twittrep-eGFP); f: comparison of the effects of S27T-beams and S27R, S27I-beams to purified Twittrep-eGFP (S27T 1-3: three experiments for the regeneration of S27T-beams purified Twittrep-eGFP; three experiments for the regeneration of S27R 1-3: S27R-beams purified Twittrep-eGFP; three experiments for the regeneration of S27I 1-3: S27I-beams purified Twittrep-eGFP); g: comparison of effects of S27T and S27C Beads purified Twittrep-eGFP (three experiments with S27T1-3: S27T Beads purified Twittrep-eGFP; three experiments with S27C 1-3: S27C Beads purified Twittrep-eGFP); h: comparison of effects of S27T Beads and S27V Beads purified Twitepp-eGFP (three experiments on the regeneration of S27T1-3: S27T Beads purified Twitepp-eGFP; three experiments on the regeneration of S27 1-3: S27C Beads purified Twitepp-eGFP)).

FIG. 3 shows the results of the enrichment of the mutant-related protein with the Twist (A: S27A Beads to purify the Twist-eGFP and regenerate six experiments), B: S27C Beads to purify the Twist-eGFP and to reuse the map (1-6: S27C Beads to purify the Twist-eGFP and regenerate six experiments), C: S27G Beads to purify the Twist-eGFP and to reuse the map (1-6: S27G Beads to purify the Twist-eGFP and to regenerate six experiments), D: S27Q Beads to purify the Twist-eGFP and to reuse the map (1-6: S27Q Beads to purify the Twist-eGFP and to regenerate six experiments), E: S27K Beads to reuse the GFP-GFP and to reuse the map (1-6: S27B to purify the Twist-6: bead-27B to purify the Twist-27B), C: S27G Beads to reuse the Twist-GFP and to reuse the map (1-6: S27A Beads to purify the Twist-GFP and to regenerate six experiments), D: S27P to reuse the map (1-6: S27B Beads to purify the Twist-22).

FIG. 4 shows the single results of the effect of the 27-phase mutant on Strep-eGFP (A: S27T, S27A, S27V, S27P, S27L, S27H, S27G beans on single comparison of the effect of the Strep-eGFP on purification; B: S27T, S27I, S27M, S27Q, S27C, S27K beans on single comparison of the effect of Strep-eGFP on purification).

FIG. 5 is a comparison of the enrichment of the mutant at position 27 with Bio-eGFP and Bio-BSA (A: S27T Beads and S27A, S27V, S27L, S27H, S27G Beads purification enrichment of Bio-eGFP and Bio-BSA comparison (In: total protein added; S27T the protein eluted from S27T tags; S27A, eluting the eluted proteins from S27A Beads; S27V-S27V Beads eluted proteins, S27L-S27L Beads eluted proteins, S27H-S27G-Beads eluted proteins, B-S27T Beads and S27I, S27M, S27Q, S27C, S27K Beads purification enrichment Bio-eGFP and Bio-BSA comparison (In: total amount of added proteins; S27T-S27T Beads eluted proteins; S27I-S27I Beads eluted proteins; S27M-S27Q-S27C-S27C Beads eluted proteins; S27C-S-S, S-and, and,.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

Example 1, screening of the 27-position mutant enriched in Twoinstrep modified proteins

In order to improve the binding capacity of Streptavidin and Strep tag II, reduce the binding capacity of Strep tag and Biotin (Biotin), synthesize the binding site of Strep tag and its ligand, and make site-directed mutation on key amino acids forming hydrogen bond between Strep tag and Biotin, so that it can retain the original product advantages, and can make up for the deficiency, and can weaken the binding of Biotin and Strep tag so as to obtain Strep tag mutant (Strep tag muts) capable of reversibly binding with Biotin. The 27 th amino acid is subjected to other amino acid mutations, which are S27T, S27G, S27A, S27C, S27D, S27E, S F, S27H, S27I, S27K, S27L, S27M, S27N, S27Q, S27R, S V, S27Y, S27P, S W, respectively, and the mutated protein sequences are respectively shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38 and SEQ ID NO.40; the gene sequences are respectively shown as SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37 and SEQ ID NO.37.

EXAMPLE 2 preparation of streppnectin mut

1) Taking 5 mu L of extracted plasmid containing streppatin mut designed as above (the plasmid containing streppatin mut takes PIISA-His-streppatin as a template, a primer is designed to carry out site-directed mutagenesis on the plasmid, PIISA-His-streppatin is synthesized by a company, PIISA-His-streppatin is shown as SEQ ID NO. 45) and added into 100 mu L of BL21 code plus (DE 3) competent cells, after ice bath for 30min, heat shock is carried out for 90s at 42 ℃, then the plasmid is kept stand on ice for 2min, 900 mu L of LB culture medium is added into a shaking table at 37 ℃ and 200rpm for 1h, the plasmid is coated on an ammonia-resistant flat plate containing 100 mu g/mL, and the plasmid is cultured overnight at the constant temperature of 37 ℃;

2) The next day, a single colony was picked up from the overnight culture plate to 10mL of LB medium containing 100. Mu.g/mL ampicillin, cultured in a shaker at 37℃and 200rpm for 12 hours, 10mL of the cultured bacterial liquid was transferred to 1L of LB medium containing 100. Mu.g/mL ampicillin, cultured in a shaker at 37℃and 200rpm, and the bacterial liquid was cultured as OD ₆₀₀ Cooling the bacterial liquid to 0 ℃ when the bacterial liquid reaches 1.5; adding IPTG with the final concentration of 1mM into the cooled bacterial liquid, and culturing at 16 ℃ and 220rpm for 18 hours; after the cultivation is finished, all the escherichia coli is collected by centrifugation at 3500rpm for 20min at 4 ℃ in a high-capacity low-temperature centrifuge, all the supernatant is poured out, the escherichia coli is resuspended in 25ml of 50mM PBS (pH 7.4) buffer solution, and PMSF with the final concentration of 1mM is added;

3) Crushing the resuspended escherichia coli by using an ultrasonic crusher, performing ultrasonic treatment at a low temperature for 3 seconds at 40% of power, stopping for 7 seconds, performing ultrasonic treatment for 20 minutes, heating the ultrasonic bacterial liquid at 60 ℃ for 15 minutes, centrifuging at 4 ℃ at 15000rpm for 20 minutes, taking the supernatant, performing suction filtration on the supernatant by using a 0.45 mu m filter membrane into a clean 50mL centrifuge tube, and placing the supernatant on ice;

4) Equilibrating the treated Ni-IDA beads with 50mL 50mM PBS (pH 7.4) buffer solution, adding the supernatant of the escherichia coli subjected to suction filtration in the previous step after the equilibration, collecting lysate flowing out of the column, and repeating loading;

5) After loading, the Ni column was rinsed with 50mM PBS (pH 7.4) containing 5mM imidazole, totaling 100mL;

6) The Ni column was washed with 50mM PBS (pH 7.4) buffer containing 40mM imidazole, totaling 50mL;

7) Eluting the target protein with 20mL of 50mM PBS (pH 7.4) buffer containing 250mM imidazole, and placing the eluted protein on ice after the elution is finished;

8) Adding 8.72g of ammonium sulfate into the eluted protein, oscillating to dissolve the ammonium sulfate, standing on ice for 30min after the ammonium sulfate is dissolved, and precipitating His-streppTactin mut protein;

9) Centrifuging the protein from the previous step at 15000rpm at 4deg.C for 10min, discarding supernatant after centrifugation, precipitating with 2mL of 10mM NaHCO containing 5mM EDTA ₃ The buffer was dissolved and the dissolved protein was centrifuged again at 15000rpm at 4℃for 10min, and the supernatant was retained after centrifugation.

Example 3 Cross-linking immobilization of Streptactin mut

1) Taking 2mL of purified His-streppTactin mut protein, placing the protein in 2L of 200mM NaHCO ₃ Dialyzing in 500mM NaCl buffer solution, and replacing the dialyzate once every 2 hours, wherein the dialyzate is replaced twice in total;

2) The His-streppTactin mut protein after dialysis is measured for ultraviolet absorption value at 280nm wavelength, and the protein concentration and the total protein mass after dialysis are calculated according to the protein absorption value of 2.84 per milligram;

3) Crosslinking was immobilized at a level of 12mg StrepTactin mut protein per ml of NHS microsphere (NHS Beads), the NHS Beads were activated with 5 times the volume of 1mM HCl solution, and after activation, 200mM NaHCO was used at 5 times the volume of Beads ₃ Balancing NHS Beads by using 500mM NaCl buffer solution, adding dialyzed protein after balancing, and rotating and crosslinking for 12 hours at 4 ℃;

4) After the end of the cross-linking, the Beads were washed once with 5 volumes of 100mM Tris-HCl (pH 8.5) buffer, and unreacted NHS groups on the Beads were blocked with 5 volumes of 100mM Tris-HCl (pH 8.5) buffer, and the blocking was performed at 4℃for 12 hours;

5) After blocking, the Beads were washed once with 5 volumes of 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA buffer, and the crosslinked streppnectin mut was stored in one volume of 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 0.03% NaN ₃ In buffer, stored at 4 ℃.

Example 4 expression of TwainStrep-eGFP and lysate preparation

1) Taking 5 mu L of extracted plasmid containing Twoin strep-eGFP, wherein the nucleotide sequence of the Twoin strep-eGFP is shown as SEQ ID NO.13 (the Twoin strep-eGFP plasmid is digested by pIHSA-Twoin strep plasmid with restriction enzyme BsaI, the eGFP is connected with a carrier through BsaI digestion site by using T4 ligase, the pIHSA-Twoin strep plasmid is obtained by connecting amplified Twoin strep sequence into BsaI digestion site of pIHSA), respectively adding the plasmid into 100 mu L of BL21 codon plus (DE 3) competent cells, carrying out heat shock at 42 ℃ for 90s after 30min on ice, standing for 2min on ice, adding 900 mu L of LB culture medium at 37 ℃ and 200rpm for 1h, coating the plasmid on a 100 mu g/mL ammonia-resistant flat plate, and culturing overnight at 37 ℃ in a shaking table at constant temperature;

2) The next day, a single colony was picked up from the overnight culture plate to 10mL of LB medium containing 100. Mu.g/mL ampicillin, cultured in a shaker at 37℃and 200rpm for 12 hours, 1mL of the bacterial liquid was transferred from 10mL of the bacterial liquid after the culture to 100mL of LB medium containing 100. Mu.g/mL ampicillin, cultured in a shaker at 37℃and 200rpm, and the culture was continued until the OD was reached ₆₀₀ Cooling the bacterial liquid to 25 ℃ when the bacterial liquid reaches 0.6; adding IPTG with the final concentration of 1mM into the cooled bacterial liquid, and culturing at 25 ℃ and 220rpm for 10 hours; after the cultivation is finished, all the escherichia coli is collected by centrifugation at 3500rpm for 20min at 4 ℃ in a high-capacity low-temperature centrifuge, all the supernatant is poured out, the escherichia coli is resuspended in 10mL of 50mM Tris-HCl (pH 7.4) and 150mM NaCl buffer, and PMSF with the final concentration of 1mM is added;

3) Crushing the resuspended escherichia coli by using an ultrasonic crusher, performing ultrasonic treatment at a low temperature for 3 seconds at 40% of power, stopping for 7 seconds, performing ultrasonic treatment for 5 minutes, centrifuging the ultrasonic bacterial liquid at 4 ℃ and 15000rpm for 20 minutes, taking a supernatant, performing suction filtration on the supernatant by using a 0.45 mu m filter membrane into a clean 15mL centrifuge tube, and placing the supernatant on ice; to obtain TwainStrep-eGFP lysate.

Examples 4, strepTactin mut Beads purification of TwainStrep-eGFP

1) Mu. L StrepTactin mut Beads, equilibrated with 200. Mu.L of 20mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM PMSF buffer, centrifuged at 3000rpm for 1min at 4℃and the supernatant discarded;

2) 100. Mu.L of the prepared TwainStrep-eGFP lysate is added into the bands immobilized by stretTactin mut crosslinking, and the bands are combined by rotating at 4 ℃ for 30min;

3) After completion of binding, the beads were washed 3 times with 200. Mu.L of 20mM Tris-HCl (pH 8.0), 150mM NaCl, 1mM EDTA, 0.5% Triton-X100 buffer, and the beads were washed 2 times with 200. Mu.L of 20mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA buffer, 5 minutes each time, and then centrifuged at 3000rpm at 4℃for 1 minute, and the supernatant was discarded;

4) Eluting protein on the beads with 20 μL of 50mM Tris-HCl (pH 7.4), 150mM NaCl and 5mM Biotin buffer, adding the eluent into the beads, standing for 5min, centrifuging at 3000rpm for 1min at 4 ℃, collecting the separated liquid, and eluting for 3 times;

5) At the end of purification, samples from each step were sampled, 5X SDS Loading Buffer (containing DTT) was added, and after heating at 95℃for 5min, 16.5% polypropylene gel was electrophoresed and stained with Coomassie brilliant blue, the results are shown in FIG. 1. The results showed that S27T, S27G, S27A, S27C, S27F, S27H, S27I, S27K, S27L, S27M, S27N, S27Q, S27R, S27V, S P was comparable to the Twinstrep single junction enrichment capability. In this example, elution was performed with a buffer containing 5-10mM Biotin.

2. StrepTactin mut Beads purification of TwainStrep-eGFP and regeneration thereof

1) 200 mu L of 20mM Tris-HCl (pH 7.4), 150mM NaCl and 1mM EDTA buffer solution are added into 15 mu L StrepTactin mut Beads of purified Twainstrep-eGFP to wash beads for 3 times, each time for 5min, and then the beads are centrifuged at 4 ℃ and 3000rpm for 1min, the supernatant is discarded, and StrepTactin mut Beads can be reused after 3 times of washing;

2) The purification effect of StrepTactin mut Beads after regeneration was tested according to the purification procedure of StrepTactin mut Beads for Twainstrep-eGFP, the purification was completed and regenerated according to the regeneration procedure, and the procedure was repeated 3 times, and after the completion, the sample was sampled for each purification, 5X SDS Loading Buffer (containing DTT) was added, and after heating at 95℃for 5min, 16.5% polypropylene gel electrophoresis was performed, and the result was stained with Coomassie brilliant blue, as shown in FIG. 2. S27T and W120H were regenerated 6 times or more as described above, and after the completion of the regeneration, each purified sample was sampled, 5X SDS Loading Buffer (containing DTT) was added thereto, and after heating at 95℃for 5 minutes, 16.5% polypropylene gel was subjected to electrophoresis, and stained with Coomassie Brilliant blue, and the results were shown in FIG. 3. In this example, the washing with PBS buffer or TE buffer can be used again, so that the range of the adaptation buffer is wide.

The above results indicate that the binding enrichment ability of threonine (Thr) (S27T), alanine (Ala) (S27A), valine (Val) (S27V), asparagine (Asn) (S27N), methionine (Met) (S27M), proline (Pro) (S27P), lysine (Lys) (S27K), cysteine (Cys) (S27C), isoleucine (Ile) (S27I) and the like to Twinstrep is equivalent; when 27 is mutated to glycine (Gly) (S27G), the protein load for enriching the Twinstrep tag for the first time is higher, the load is reduced after regeneration but the reproducibility is still better than strepptactin. The mutant was subjected to mutation at position 27 to phenylalanine (Phe) (S27F), glutamine (Gln) (S27Q), histidine (His) (S27H), arginine (Arg) (S27R), leucine (Leu) (S27L), and the enriched Twittrep load was relatively reduced, but the mutant was relatively easy to regenerate, and after elution, the mutant was washed with Tri-HCl buffer, PBS buffer or TE for reuse, while Streptatin had little enrichment capacity for Twittrep fusion protein if it was simply regenerated with Tri-HCl (pH 7.4) buffer.

Mutation at position 27 into the acidic amino acids aspartic acid (Asp) (S27D) and glutamic acid (Glu) (S27E) and tyrosine (Tyr) (S27Y), tryptophan (Trp) (S27W) significantly reduced the binding to Biotin, especially mutation into D and E reduced no enrichment to Twostrep.

Example 5, screening of the 27-position mutant enriched Strep-modified proteins

And carrying out protein enrichment test on the protein with Strep label on the mutants which are screened out and have better protein enrichment effect on the protein with the Twittrep label. The protein amino acid sequence of Strep tag is shown as SEQ ID NO.41, and the preparation method is the same as that of Twainstrep-eGFP, except that the nucleotide sequence shown as SEQ ID NO.42 is connected into Strep-eGFP. The results are shown in FIG. 4. The results show that the loading amount of the S27A on the protein with the Strep label is higher, and the enrichment effect of the S27T, S27C, S27P, S27H, S27I, S27K and S27V on the protein with the Strep label is relatively obvious.

Example 6, screening of mutant enriched biotin-modified proteins at position 27

1. Expression of biotin-modified eGFP (Bio-eGFP) and lysate preparation

Biotin can be specifically modified on lysine residues in the Avi tag by the action of biotin ligase (BirA) to produce a biotinylated Avi tag.

1) Taking 5 mu L of extracted plasmid containing CBD-BirA, wherein the CBD-BirA gene sequence is shown as SEQ ID NO.43 (the CBD-BirA plasmid is cut by restriction enzymes XhoI and NcoI and PET28a-CBD is connected by T4 ligase through the XhoI cutting site, the CBD of PET28a-CBD is connected with PET28a carrier by BSAI cutting site) and plasmid containing Avi-eGFP, the Avi-eGFP gene sequence is shown as SEQ ID NO.44 (the plasmid containing Avi-eGFP is cut by restriction enzymes BamHI and XhoI and PET22b-Avi plasmid is connected by T4 ligase through BamHI and XhoI cutting site, the Avi is connected with PET22b by recombination), then adding the plasmid into 100 mu L of BL21 codon plus (DE 3) competent cells, carrying out heat shock 90s at 42 ℃ after 30min of ice, then adding the plasmid containing Avi-eGFP into 2 ℃ and standing at 37 mu L of ice shaking table at a constant temperature of 200 mu.L, and standing overnight at 37 mu.L of shaking table for 50 mu.m/mL;

2) The following day, a single colony was picked from the overnight culture plate into 10mL of LB medium containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin, cultured in a shaker at 37℃and 200rpm for 12 hours, transferred from 10mL after the culture into 1L of LB medium containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin, cultured in a shaker at 200rpm at 37℃and cultured as OD ₆₀₀ Cooling the bacterial liquid to 25 ℃ when the bacterial liquid reaches 0.6; adding IPTG with the final concentration of 1mM and Biotin with the final concentration of 1mM into the cooled bacterial liquid, and culturing at 25 ℃ and 220rpm for 10 hours; after the cultivation is finished, all the escherichia coli is collected by centrifugation at 3500rpm for 20min at 4 ℃ in a high-capacity low-temperature centrifuge, all the supernatant is poured out, the escherichia coli is resuspended in 30mL of 50mM Tris-HCl (pH 7.4) and 150mM NaCl buffer, and PMSF with the final concentration of 1mM is added;

3) Crushing the resuspended escherichia coli by using an ultrasonic crusher, performing ultrasonic treatment at a low temperature for 3 seconds at 40% of power, stopping for 7 seconds, performing ultrasonic treatment for 20 minutes, centrifuging at a temperature of 4 ℃ and at a speed of 15000rpm for 20 minutes, taking a supernatant, performing suction filtration on the supernatant by using a filter membrane of 0.45 mu m to a clean 50mL centrifuge tube, and placing the supernatant on ice to obtain the Bio-eGFP lysate.

Preparation of Bio-BSA, dissolving 664.5mg of BSA protein in buffer, weighing 34.1mg of Biotin-NHS into dissolved BSA, reacting overnight at 25℃and dialyzing to 200mM NaHCO ₃ 500mM NaCl buffer. The mutants selected above were subjected to enrichment tests on Bio-BSA and Bio-eGFP, and the results are shown in FIG. 5. The results show that the enrichment effect of S27T, S, M, S, C, S, K, S, A, S, V, S, I, S, P, S, H, S and 27G is obvious. The mutants can be used for enrichment and purification of Strep Tag II and also can be used for enrichment and purification of biotin modified proteins.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A mutant protein S27L of streptavidin having a serine mutation at position 27, characterized in that: the mutant protein is formed by taking amino acid residues 44 to 47 of wild-type streptavidin as VTAR, and taking serine mutation at 27 th position of the wild-type streptavidin as leucine, and is marked as S27L; the amino acid sequence is shown as SEQ ID NO. 24.

2. The immobilized complex of a mutein of serine mutation at position 27 of streptavidin and a microsphere according to claim 1.

3. Use of a mutant protein according to the serine mutation at position 27 of streptavidin or of a immobilization complex according to claim 2 for purification of Strep tag ii or of a Twinstrep tag protein.

4. Use of a mutant protein having a serine mutation at position 27 of streptavidin according to claim 1 or a cross-linked complex according to claim 2 for purifying a biotin-modified protein.

5. A method for purifying Strep tag ii or Twinstrep tag proteins using the mutant protein of serine mutation at position 27 of streptavidin according to claim 1, wherein: lysates expressing proteins containing Strep tag II or Twittrep tag were combined with the immobilized complex of the mutein, washed with buffer, and finally eluted with 5-10mM Biotin buffer, and the eluate was collected.

6. The method for purifying Strep tag II or Twainstep tag protein by using the mutant protein of the serine mutation at position 27 of streptavidin according to claim 5, wherein the method comprises the steps of: the Biotin buffer solution also comprises the following steps of recycling after elution: and adding buffer solution into the immobilized complex purified by Strep tag II or Twittrep tag protein for washing, and then reusing.