The mutant protein S27L, derived from a serine mutation at position 27 of streptavidin, and its applications.

Abstract

This invention discloses the mutant protein S27L, which has a serine mutation at position 27 of streptavidin, and its applications. The mutant protein has amino acids VTAR at positions 44-47 of wild-type streptavidin, and the serine at position 27 of wild-type streptavidin is mutated to a different amino acid. The mutant protein has increased binding affinity to Strep tag II, improving its purification ability for single Strep and TwinStrep fusion proteins. At the same time, its binding affinity to biotin is weakened, making the interaction between biotin and the protein reversible. After elution, it can be washed and regenerated for reuse. Therefore, it can be better used for the purification of biotin-modified proteins and Strep tag II-tagged proteins.

Description

Technical Field

[0001] This invention relates to a streptavidin-mutated mutant protein that, after mutation, can reversibly bind biotin, and therefore can be used for protein purification, regeneration, and reuse. Background Technology

[0002] Streptavidin exhibits a strong non-covalent binding affinity to biotin. The dissociation equilibrium constant K of wild-type streptavidin binding to biotin is... d In 10 -14 At mol / L, streptavidin represents the strongest known non-covalent interaction in nature, thus finding wide application in molecular biology, including affinity chromatography, live-cell fluorescence imaging, proteomics, and the immobilization of biotinylated enzymes. While streptavidin has broad applications, each specific application demands more refined properties from it. For example, affinity chromatography requires low affinity and a high dissociation constant to effectively elute target molecules from streptavidin-containing microspheres. Because wild-type streptavidin binds very strongly to biotin-modified proteins, it requires very harsh conditions, such as heating at 95°C in a buffer solution containing a high concentration of biotin, to elute some of the protein. Therefore, it cannot be used for non-denaturing affinity chromatography of biotin-modified proteins. The affinity purification application of wild-type streptavidin utilizes its strong affinity for the 38-amino acid peptide SBP (amino acid sequence MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), with a dissociation equilibrium constant K... d 10 -9 SBP peptides were fused to the N-terminus or C-terminus of the target protein using a concentration of mol / L as an affinity purification tag, followed by competitive elution with biotin. However, this application suffers from a drawback: the extremely strong binding affinity of biotin to wild-type streptavidin prevents effective regeneration of wild-type streptavidin, thus limiting the application of SBP tags. To expand the application range of streptavidin, it is necessary to modify streptavidin to weaken its binding affinity to biotin.

[0003] Voss et al. mutated amino acids 44-47 of streptavidin to obtain the StrepTactin mutant (amino acids 44-47 have the following sequence: Val-Thr-Ala-Arg, a truncated sequence from positions 15 to 139 of wild-type streptavidin). This mutant specifically binds to the short peptide Strep tag II (peptide sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). The dissociation equilibrium constant K for Strep tag II binding is... dIn 10 -7 mol / L, but its dissociation equilibrium constant K for biotin is... d Still in 10 -11 ~10 -12 The concentration of mol / L makes its binding with biotin irreversible. Wong et al. mutated serine (Ser) at position 27 of wild-type streptavidin to alanine (Ala) (S27A) and glycine (Gly) at position 48 to threonine (Thr) (G48T) to obtain the SAVSBPM18 mutant. The dissociation equilibrium constant K of this mutant with biotin was... d Increase by 10 -8 The concentration of mol / L makes the binding of Biotin to SAVSBPM18 reversible.

[0004] Due to the relatively long length of SBP tags, their applications are limited. Currently, the most widely used combination is with StrepTactin and Strep tags. However, the binding strength between Strep tag II alone and StrepTactin is still insufficient. Therefore, in practical applications, two Strep tag IIs are tandemly linked (i.e., Twinstrep tags, peptide sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(Gly-Gly-Gly-Ser)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, whose encoded nucleotides are shown in SEQ ID NO.1, and amino acid sequence is shown in SEQ ID NO.2) to achieve the dissociation equilibrium constant K. d Reaching 10 -9 The concentration of mol / L significantly enhances its binding affinity. However, when used to purify StrepTactin fusion proteins tagged with Strep tag II, it still has the following drawbacks. In particular, elution with biotin makes StrepTactin difficult to regenerate and reuse, and regeneration with denaturing agents, strong acids, or strong bases reduces its lifespan. When eluting the target protein, the more expensive desulfurized biotin is often used instead of biotin (Schmidt et al., 2007). However, because the price of desulfurized biotin is much higher than that of biotin, the concentration used is higher, which increases the cost of use and is not conducive to industrial production. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a mutant protein in which amino acids 44-47 of wild-type streptavidin are VATR and serine at position 27 is mutated; a second objective of the present invention is to provide a fixed complex containing the mutant protein containing the serine at position 27 of the streptavidin; a third objective of the present invention is to provide the application of the mutant protein containing the serine at position 27 of the streptavidin or the fixed complex in purifying Strep tag II or Twinstrep-tagged proteins; a fourth objective of the present invention is to provide the application of the mutant protein containing the streptavidin or the fixed complex in purifying biotin-modified proteins; and a fifth objective of the present invention is to provide a method for purifying Strep tag II or Twinstrep-tagged proteins using the mutant protein containing the serine at position 27 of the streptavidin.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. The mutant protein S27L, which is a serine mutation at position 27 of streptavidin, wherein the mutant protein is wild-type streptavidin with amino acid residues VTAR from position 44 to 47, and a serine mutation at position 27 of wild-type streptavidin with leucine, is denoted as S27L; the amino acid sequence is shown in SEQ ID NO.24.

[0008] 2. The immobilized complex of the streptavidin protein with a serine mutation at position 27 and the microspheres.

[0009] 3. The application of the streptavidin protein with a serine mutation at position 27 or the immobilization complex in the purification of Streptag II-tagged or Twinstrep-tagged proteins.

[0010] 4. The application of the streptavidin protein with a serine mutation at position 27 or the cross-linked complex in the purification of biotin-modified proteins.

[0011] 5. A method for purifying Strep tag II or Twinstrep tag protein using the mutant protein with the 27th serine mutation of streptavidin: The lysate expressing the Strep tag II or Twinstrep tag protein is combined with the immobilized complex of the mutant protein, washed with buffer, and finally eluted with 5-10 mM Biotin buffer, and the eluent is collected.

[0012] The preferred embodiment of the present invention is a method for purifying Strep tag II or Twinstrep-tagged proteins from streptavidin with a serine mutation at position 27, characterized in that: after elution with Biotin buffer, the protein is further recycled, specifically by adding buffer to the purified Strep tag II or Twinstrep-tagged protein immobilized complex for washing and reuse.

[0013] The beneficial effects of this invention are as follows: This invention provides a mutant protein with a serine mutation at position 27 of the streptavidin. The serine (Ser) at position 27 of wild-type streptavidin is mutated into a mutant with different amino acids. The mutant protein has increased binding affinity to Strep tag II, which improves its purification ability for Strep and TwinStrep fusion proteins. At the same time, the binding affinity to biotin is weakened, making the interaction between biotin and the protein reversible. After elution, the protein can be washed and regenerated for reuse. Therefore, it can be better used for the purification of biotin-modified proteins and Strep tag II-tagged proteins. Biotin is used to elute biotin-modified proteins, and after elution, StrepTactin mut is washed and regenerated with buffer for reuse. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0015] Figure 1 Screening for enrichment of Twinstrep-modified proteins for 27 related mutants (A: Single-shot comparison of the purification effect of StepTactin Beads and S27T, S27A, S27G, S27E, S27Q, S27W, S27C, S27H Beads on Twinstrep-eGFP; B: Single-shot comparison of the purification effect of StepTactin Beads and S27T, S27N, S27V, S27M, S27P, S27L, S27K Beads on Twinstrep-eGFP; C: Single-shot comparison of the purification effect of StepTactin Beads and S27T, S27Y, S27I, S27D, S27PR, S27F Beads on Twinstrep-eGFP).

[0016] Figure 2Results of three regeneration experiments to enrich Twinstrep-modified proteins for 27 related mutants (A: Comparison of the purification effects of S27T Beads and S27A, S27G Beads on Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27A 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27A Beads; S27G 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27G Beads)); B: Comparison of the purification effects of S27T Beads and S27H, S27Q Beads on Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27H 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27H) Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27Q1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27Q Beads; C: Comparison of the effects of S27T Beads and S27L, S27P Beads in purifying Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27L 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27L Beads; S27P 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27P Beads); D: Comparison of the effects of S27T Beads and S27M, S27F Beads in purifying Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27M... 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27M Beads; 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27F Beads; E: Comparison of the effects of S27T Beads and S27N, S27K Beads on the purification of Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27N 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27N Beads; S27K 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27K Beads).F: Comparison of the purification effects of S27T Beads and S27R, S27I Beads on Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27R 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27R Beads; S27I 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27I Beads); G: Comparison of the purification effects of S27T Beads and S27C Beads on Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27TB Beads; S27C 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27C Beads); H: S27T Beads and S27V Comparison of the effects of bead purification of Twinstrep-eGFP (S27T 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27T Beads; S27 1-3: Three regeneration experiments of Twinstrep-eGFP purified by S27C Beads)).

[0017] Figure 3Results of six regeneration experiments to enrich Twinstrep-modified proteins for 27 related mutants (A: Reuse of purified Twinstrep-eGFP using S27A Beads (1-6: Experiments of purified Twinstrep-eGFP using S27A Beads and six regeneration experiments); B: Reuse of purified Twinstrep-eGFP using S27C Beads (1-6: Experiments of purified Twinstrep-eGFP using S27C Beads and six regeneration experiments); C: Reuse of purified Twinstrep-eGFP using S27G Beads (1-6: Experiments of purified Twinstrep-eGFP using S27G Beads and six regeneration experiments); D: Reuse of purified Twinstrep-eGFP using S27Q Beads (1-6: Experiments of purified Twinstrep-eGFP using S27Q Beads and six regeneration experiments); E: S27K... Figure 1: Reuse of purified Twinstrep-eGFP using S27K Beads (1-6: Experiments of purifying Twinstrep-eGFP using S27K Beads and regenerating it six times); Figure 2: Reuse of purified Twinstrep-eGFP using S27L Beads (1-6: Experiments of purifying Twinstrep-eGFP using S27L Beads and regenerating it six times); Figure 3: Reuse of purified Twinstrep-eGFP using S27M Beads (1-6: Experiments of purifying Twinstrep-eGFP using S27M Beads and regenerating it six times); Figure 4: Reuse of purified Twinstrep-eGFP using S27P Beads (1-6: Experiments of purifying Twinstrep-eGFP using S27P Beads and regenerating it six times); Figure 5: Reuse of purified Twinstrep-eGFP using S27V Beads (1-6: Experiments of purifying Twinstrep-eGFP using S27V Beads and regenerating it six times)

[0018] Figure 4 Single-shot results of the purification effect of Strep-eGFP for 27 related mutants (A: Single-shot comparison of the purification effect of S27T, S27A, S27V, S27P, S27L, S27H, S27G Beads; B: Single-shot comparison of the purification effect of S27T, S27I, S27M, S27Q, S27C, S27K Beads).

[0019] Figure 5Comparison of Bio-eGFP and Bio-BSA enrichment for 27 related mutants (A: Comparison of Bio-eGFP and Bio-BSA enrichment after purification using S27T Beads and S27A, S27V, S27L, S27H, S27G Beads (In: Total amount of protein added; S27T: Protein eluted from S27T Beads; S27A: Protein eluted from S27A Beads; S27V: Protein eluted from S27V Beads; S27L: Protein eluted from S27L Beads; S27H: Protein eluted from S27H Beads; S27G: Protein eluted from S27G Beads)); B: S27T Beads and S27I, S27M, S27Q, S27C, S27K) Beads purification and enrichment comparison of Bio-eGFP and Bio-BSA (In: total amount of protein added; S27T: protein eluted from S27T Beads; S27I: protein eluted from S27I Beads; S27M: protein eluted from S27M Beads; S27Q: protein eluted from S27Q Beads; S27C: protein eluted from S27C Beads; S27K: protein eluted from S27K Beads). Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Example 1: Screening for enrichment of Twinstrep-modified proteins by mutants associated with position 27

[0022] In this invention, to enhance the binding affinity of streptavidin to Strep Tag II and reduce the binding affinity of StrepTactin to biotin, a site-directed mutation is performed on the key amino acids that form hydrogen bonds between StrepTactin and biotin, taking into account the binding sites of StrepTactin and its ligands. The aim is to retain the advantages of the original product while compensating for its shortcomings, weakening the binding of biotin to StrepTactin, and thus obtaining StrepTactin mutants that can reversibly bind to biotin. The 27th position was mutated by various other amino acid mutations, namely S27T, S27G, S27A, S27C, S27D, S27E, S27F, S27H, S27I, S27K, S27L, S27M, S27N, S27Q, S27R, S27V, S27Y, S27P, and S27W. The resulting protein sequences are shown in 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. NO.40; the gene sequences are respectively 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.

[0023] Example 2: Preparation of StrepTactin mut

[0024] 1) Take 5 μL of the plasmid containing the StrepTactin muts designed above (the plasmid containing StrepTactin muts is designed with primers to perform site-directed mutagenesis using PIISA-His-StrepTactin as a template. PIISA-His-StrepTactin is synthesized by the company and is listed as SEQ ID NO.45) and add it to 100 μL of BL21 codon plus (DE3) competent cells. After incubating on ice for 30 min, heat shock at 42℃ for 90 s, then stand on ice for 2 min. Add 900 μL of LB medium and revive in a shaker at 37℃ and 200 rpm for 1 h. Spread the revival medium on a plate containing 100 μg / mL ampicillin and incubate overnight at 37℃.

[0025] 2) On the second day, pick a single colony from the overnight plate and transfer it to 10 mL of LB medium containing 100 μg / mL ampicillin. Incubate at 37°C and 200 rpm for 12 h. Transfer the resulting 10 mL of culture to 1 L of LB medium containing 100 μg / mL ampicillin and incubate at 37°C and 200 rpm. When the OD... 600 When the concentration reaches 1.5, the bacterial culture is cooled to 0°C; the cooled bacterial culture is then incubated with 1 mM IPTG at 16°C and 220 rpm for 18 h; after the incubation is completed, all E. coli are collected by centrifuging at 3500 rpm for 20 min at 4°C in a large-capacity low-temperature centrifuge, all supernatant is discarded, and the E. coli are resuspended in 25 mL of 50 mM PBS (pH 7.4) buffer, and then PMSF is added to a final concentration of 1 mM.

[0026] 3) The resuspended E. coli was disrupted using an ultrasonic disruptor. Under low temperature conditions, the ultrasonication was performed at 40% power for 3 seconds, followed by a 7-second pause, for a total of 20 minutes. The ultrasonicated bacterial solution was then heated at 60°C for 15 minutes. After heating, the bacterial solution was centrifuged at 4°C and 15,000 rpm for 20 minutes. The supernatant was collected and filtered through a 0.45 μm filter membrane into a clean 50 mL centrifuge tube, which was then placed on ice.

[0027] 4) Equilibrate the Ni-IDA beads with 50 mL of 50 mM PBS (pH 7.4) buffer. After equilibration, add the E. coli supernatant filtered in the previous step, collect the lysis buffer flowing out of the column, and repeat the loading once.

[0028] 5) After loading the sample, wash the Ni column with 50mM PBS (pH 7.4) containing 5mM imidazole, for a total wash of 100mL;

[0029] 6) Rinse the Ni column with 50mM PBS (pH 7.4) buffer containing 40mM imidazole, for a total of 50mL;

[0030] 7) Elute the target protein with 20 mL of 50 mM PBS (pH 7.4) buffer containing 250 mM imidazole. After elution, place the eluted protein on ice.

[0031] 8) Add 8.72 g of ammonium sulfate to the eluted protein, shake to dissolve the ammonium sulfate, and place on ice for 30 min to precipitate His-StrepTactin mut protein;

[0032] 9) Centrifuge the protein from the previous step at 4°C, 15000 rpm for 10 min. After centrifugation, discard the supernatant and dissolve the precipitate in 2 mL of 10 mM NaHCO3 buffer containing 5 mM EDTA. Centrifuge the dissolved protein again at 4°C, 15000 rpm for 10 min. After centrifugation, retain the supernatant.

[0033] Example 3: Crosslinking and Immobilization of StrepTactin mut

[0034] 1) Take 2 mL of purified His-StrepTactin mut protein and dialyze the protein in 2 L of 200 mM NaHCO3 and 500 mM NaCl buffer. Change the dialysate every 2 hours for a total of two dialysate changes.

[0035] 2) The absorbance of His-StrepTactin mut protein at 280 nm wavelength was measured after dialysis. The protein concentration and total protein mass after dialysis were calculated based on an absorbance of 2.84 per milligram of protein.

[0036] 3) Crosslink 12 mg of StrepTactin mut protein per milliliter of NHS microspheres (NHS Beads), activate NHS Beads with 1 mM HCl solution in 5 times the volume of Beads, equilibrate NHS Beads with 200 mM NaHCO3 and 500 mM NaCl buffer in 5 times the volume of Beads, add dialyzed protein after equilibration, and rotate crosslink at 4°C for 12 h.

[0037] 4) After crosslinking, wash the Beads once with 5 times the volume of 100mM Tris-HCl (pH 8.5) buffer, and then block the unreacted NHS groups on the Beads with 5 times the volume of 100mM Tris-HCl (pH 8.5) buffer. Rotate and block at 4°C for 12 hours.

[0038] 5) After blocking, wash the beads once with 5 times the volume of 50mM Tris-HCl (pH 7.4), 150mM NaCl, and 1mM EDTA buffer. Store the cross-linked StrepTactin mut in 1 times the volume of the beads in 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, and 0.03% NaN3 buffer at 4°C.

[0039] Example 4: Expression of TwinStrep-eGFP and Preparation of Lysis Buffer

[0040] 1) Take 5 μL of the extracted plasmid containing TwinStrep-eGFP. The nucleotide sequence of TwinStrep-eGFP is shown in SEQ ID NO.13 (the plasmid containing TwinStrep-eGFP is obtained by digesting the pIISA-TwinStrep plasmid with the restriction endonuclease BsaI, and ligating eGFP to the vector with T4 ligase through the BsaI restriction site. The pIISA-TwinStrep plasmid is obtained by ligating the amplified TwinStrep sequence into the BsaI restriction site of pIISA). Add 5 μL of the plasmid to 100 μL of BL21 codon plus (DE3) competent cells. After incubating on ice for 30 min, heat shock at 42℃ for 90 s, then stand on ice for 2 min. Add 900 μL of LB medium and revive in a shaker at 37℃ and 200 rpm for 1 h. Spread the revival medium on a plate containing 100 μg / mL ampicillin and incubate overnight at 37℃.

[0041] 2) On the second day, pick a single colony from the overnight culture plate and transfer it to 10 mL of LB medium containing 100 μg / mL ampicillin. Incubate at 37°C and 200 rpm for 12 h. Transfer 1 mL of the culture from the 10 mL culture to 100 mL of LB medium containing 100 μg / mL ampicillin and incubate at 37°C and 200 rpm. When the OD... 600 When the concentration reaches 0.6, the bacterial culture is cooled to 25°C. After cooling, IPTG is added to the bacterial culture to a final concentration of 1 mM and the culture is incubated at 25°C and 220 rpm for 10 h. After the incubation, all E. coli are collected by centrifugation at 4°C and 3500 rpm for 20 min in a large-capacity low-temperature centrifuge. All supernatant is discarded, and the E. coli are resuspended in 10 mL of 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl buffer. Then, PMSF is added to a final concentration of 1 mM.

[0042] 3) The resuspended E. coli was disrupted using an ultrasonic disruptor. Under low temperature conditions, the ultrasonication was performed at 40% power for 3 seconds, followed by a 7-second pause, and then for 5 minutes. The ultrasonicated bacterial solution was centrifuged at 4°C, 15,000 rpm for 20 minutes. The supernatant was collected and filtered through a 0.45 μm filter membrane into a clean 15 mL centrifuge tube, which was then placed on ice to obtain the TwinStrep-eGFP lysis buffer.

[0043] Example 4: Purification of TwinStrep-eGFP by StrepTactin mut Beads

[0044] 1) Take 15 μL of StrepTactin mut Beads, equilibrate the beads with 200 μL of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM PMSF buffer, centrifuge at 4 °C and 3000 rpm for 1 min, and discard the supernatant;

[0045] 2) Take 100 μL of the TwinStrep-eGFP lysis buffer prepared above and add it to the Beads immobilized by StrepTactin mut crosslinking, and rotate to bind at 4℃ for 30 min;

[0046] 3) After binding is complete, wash the beads three times with 200 μL of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 0.5% Triton-X100 buffer. Then wash the beads twice with 200 μL of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA buffer, each time for 5 min. Then centrifuge at 4 °C and 3000 rpm for 1 min and discard the supernatant.

[0047] 4) Elute the protein on the beads with 20 μL of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 5 mM Biotin buffer. After adding the eluent to the beads, let them stand for 5 min, then centrifuge at 4 °C and 3000 rpm for 1 min, collect the separated liquid, and elute 3 times.

[0048] 5) After purification, samples from each step were taken, 5×SDS Loading Buffer (containing DTT) was added, and the mixture was heated at 95℃ for 5 min before 16.5% polypropylene gel electrophoresis. The samples were then stained with Coomassie Brilliant Blue. The results are as follows: Figure 1As shown in the figure. The results show that S27T, S27G, S27A, S27C, S27F, S27H, S27I, S27K, S27L, S27M, S27N, S27Q, S27R, S27V, and S27P have comparable enrichment abilities for single-bug Twinstrep binding. In this example, elution with a buffer containing 5-10 mM Biotin is acceptable.

[0049] 2. Regeneration and reuse of StrepTactin mut Beads after purification of TwinStrep-eGFP

[0050] 1) Add 200 μL of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA buffer to 15 μL of purified TwinStrep-eGFP StrepTactin mut Beads and wash the beads 3 times for 5 min each time. Then centrifuge at 3000 rpm for 1 min at 4 °C and discard the supernatant. After 3 washes, the StrepTactin mut Beads can be used again.

[0051] 2) The purification effect of regenerated StrepTactin mut Beads was tested according to the purification steps for TwinStrep-eGFP using StrepTactin mut Beads. After purification, regeneration was performed according to the regeneration steps, repeating this process three times. Samples from each purification were taken, and 5×SDS Loading Buffer (containing DTT) was added. After heating at 95℃ for 5 min, 16.5% polypropylene gel electrophoresis was performed, followed by Coomassie Brilliant Blue staining. The results are as follows: Figure 2 As shown. Following the above method, S27T and W120H were regenerated at least 6 times. After each regeneration, samples were taken from each purification process, 5×SDS Loading Buffer (containing DTT) was added, and the mixture was heated at 95℃ for 5 min before 16.5% polypropylene gel electrophoresis. The gels were then stained with Coomassie Brilliant Blue. The results are shown below. Figure 3 As shown. Furthermore, in this embodiment, washing with PBS buffer or TE buffer can be reused, thus adapting to a wide range of buffers.

[0052] The results indicate that mutations of serine (Ser) to threonine (Thr) (S27T), alanine (Ala) (S27A), valine (Val) (S27V), asparagine (Asn) (S27N), methionine (Met) (S27M), proline (Pro) (S27P), lysine (Lys) (S27K), cysteine ​​(Cys) (S27C), and isoleucine (Ile) (S27I) have comparable enrichment capabilities for Twinstrep. When position 27 is mutated to glycine (Gly) (S27G), the initial protein loading for enriching the Twinstrep tag is higher, and the loading decreases after regeneration, but the reproducibility is still better than StrepTactin. When the 27th position is mutated to phenylalanine (Phe) (S27F), glutamine (Gln) (S27Q), histidine (His) (S27H), arginine (Arg) (S27R), or leucine (Leu) (S27L), the enrichment capacity of Twinstrep is relatively reduced, but the regeneration and utilization are relatively good. After elution, the mutant can be regenerated by washing with Tri-HCl buffer, PBS buffer, or TE buffer and reused. In contrast, StrepTactin has almost no enrichment capacity for Twinstrep fusion protein after simple regeneration with Tri-HCl (pH 7.4) buffer.

[0053] Mutating position 27 to the acidic amino acids aspartic acid (S27D) and glutamic acid (S27E), as well as tyrosine (S27Y) and tryptophan (S27W), significantly weakened the binding effect of Twinstrep to Biotin. In particular, mutations to D and E weakened the binding effect of Twinstrep to no enrichment.

[0054] Example 5: Screening for Strep-modified proteins enriched by mutants associated with position 27

[0055] Several mutants selected above that showed good enrichment effects on proteins with the Twinstrep tag were then subjected to enrichment tests on proteins with the Strep tag. The amino acid sequence of the Strep-tagged protein is shown in SEQ ID NO.41, and the preparation method is the same as that of TwinStrep-eGFP, except that the nucleotide sequence of Strep-eGFP, as shown in SEQ ID NO.42, is inserted. The results are as follows: Figure 4 As shown in the figure. The results showed that S27A had a high enrichment capacity for Strep-tagged proteins, and S27T, S27C, S27P, S27H, S27I, S27K, and S27V also had relatively significant enrichment effects on Strep-tagged proteins.

[0056] Example 6: Screening for biotin-modified proteins enriched by mutants at position 27

[0057] 1. Expression and lysis buffer preparation of biotin-modified eGFP (Bio-eGFP)

[0058] Biotin can be specifically modified onto the lysine residues in the Avi tag by the action of biotin ligase (BirA), thus generating a biotinylated Avi tag.

[0059] 1) Take 5 μL of the extracted plasmid containing CBD-BirA (CBD-BirA gene sequence is shown in SEQ ID NO.43; the CBD-BirA plasmid was digested with restriction endonucleases XhoI and NcoI on PET28a-CBD, BirA was ligated with T4 ligase through the XhoI restriction site, and PET28a-CBD was ligated with the PET28a vector through the BSAI restriction site) and the plasmid containing Avi-eGFP (Avi-eGFP gene sequence is shown in SEQ ID NO.44; the Avi-eGFP plasmid was digested with restriction endonucleases BamHI and XhoI on PET22b-avi, eGFP was ligated with T4 ligase through the BamHI and XhoI restriction sites, and avi was ligated with PET22b through recombination). Then add to 100 μL of BL21 codon. plus(DE3) competent cells were incubated on ice for 30 min, then heat-shocked at 42°C for 90 s, and then placed on ice for 2 min. 900 μL of LB medium was added and the cells were thawed in a shaker at 37°C and 200 rpm for 1 h. The cells were then spread on plates containing 100 μg / mL ampicillin and 50 μg / mL kanamycin resistance and cultured overnight at 37°C.

[0060] 2) On the second day, pick a single colony from the overnight culture plate and transfer it to 10 mL of LB medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. Incubate at 37°C and 200 rpm for 12 h. Transfer the 10 mL culture to 1 L of LB medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin, and incubate at 37°C and 200 rpm. When OD... 600 When the concentration reaches 0.6, the bacterial culture is cooled to 25°C. After cooling, 1 mM IPTG and 1 mM Biotin are added to the bacterial culture and it is incubated at 25°C and 220 rpm for 10 h. After incubation, all E. coli are collected by centrifugation at 4°C and 3500 rpm for 20 min in a large-capacity low-temperature centrifuge. All supernatant is discarded, and the E. coli are resuspended in 30 mL of 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl buffer. Then, 1 mM PMSF is added.

[0061] 3) The resuspended E. coli was disrupted using an ultrasonic disruptor. Under low temperature conditions, the ultrasonication was performed at 40% power for 3 seconds, followed by a 7-second pause, for a total of 20 minutes. The ultrasonicated bacterial solution was then centrifuged at 4°C and 15,000 rpm for 20 minutes. The supernatant was collected and filtered through a 0.45 μm filter membrane into a clean 50 mL centrifuge tube, which was then placed on ice to obtain the Bio-eGFP lysis buffer.

[0062] To prepare Bio-BSA, 664.5 mg of BSA protein was weighed and dissolved in buffer. 34.1 mg of Biotin-NHS was added to the dissolved BSA. The reaction was carried out overnight at 25°C, followed by dialyzing into a 200 mM NaHCO3, 500 mM NaCl buffer solution. The mutants selected above were then subjected to enrichment tests on Bio-BSA and Bio-eGFP. The results are as follows... Figure 5 As shown in the figure. The results indicate that S27T, S27M, S27C, S27K, S27A, S27V, S27I, S27P, S27H, and S27G showed significant enrichment effects. This suggests that these mutants can be used not only for the enrichment and purification of Strep Tag II, but also for the enrichment and purification of biotin-modified proteins.

[0063] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The mutant protein S27L, derived from a serine mutation at position 27 of streptavidin, is characterized by: The mutant protein is wild-type streptavidin with amino acid residues VTAR at positions 44 to 47, and serine at position 27 of wild-type streptavidin is mutated to leucine, denoted as S27L; the amino acid sequence is shown in SEQ ID NO.

24.

2. The immobilized complex of the streptavidin protein with a serine mutation at position 27 as described in claim 1 and the microspheres.

3. The use of the streptavidin protein with a serine mutation at position 27 as described in claim 1 or the immobilization complex as described in claim 2 in the purification of Strep tag II or Twinstrep tag proteins.

4. The use of the streptavidin protein with a serine mutation at position 27 as described in claim 1 or the immobilization complex as described in claim 2 in the purification of biotin-modified proteins.

5. A method for purifying Strep tag II or Twinstrep tag protein using the mutant protein with a serine mutation at position 27 of streptavidin as described in claim 1, characterized in that: The lysate expressing a protein containing a Strep tag II tag or a Twinstrep tag is bound to the immobilized complex of the mutant protein, washed with buffer, and finally eluted with 5-10 mM Biotin buffer. The eluent is then collected.

6. The method for purifying Strep tag II or Twinstrep tag protein from a streptavidin protein with a serine mutation at position 27 as described in claim 5, characterized in that: Biotin buffer elution also allows for regeneration, specifically by adding buffer to the purified Strep tag II or Twinstrep tag protein immobilization complex for washing and reuse.

Images