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Mutant protein with 27th serine mutation of streptavidin and application of mutant protein

A streptavidin and mutant protein technology, which is applied in the direction of peptides containing affinity tags, applications, peptide preparation methods, etc., can solve the problems of reduced service life, increased use cost, and high use concentration, so as to improve the purification ability Effect

Active Publication Date: 2022-05-13
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when StrepTactin is used to purify the fusion protein of Strep tag II tag, it still has the following disadvantages, especially after elution with biotin, it will make it difficult to regenerate StrepTactin, and regenerate it with denaturants, strong acids, strong bases, etc., reducing its use Lifespan; more expensive desthiobiotin is often used instead of biotin to elute the target protein (Schmidt et al, 2007), but because the price of desthiobiotin is much higher than that of biotin, the concentration used is higher, Therefore, the cost of use has increased, which is not conducive to industrial production.

Method used

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  • Mutant protein with 27th serine mutation of streptavidin and application of mutant protein
  • Mutant protein with 27th serine mutation of streptavidin and application of mutant protein
  • Mutant protein with 27th serine mutation of streptavidin and application of mutant protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1、27

[0024] Example 1, Screening of 27 related mutants enriched for Twinstrep modified proteins

[0025] In the present invention, in order to improve the binding ability of streptavidin (Streptavidin) and Strep tag II, reduce the binding ability of StrepTactin and biotin (Biotin), synthesize the binding site of StrepTactin and its ligand, form hydrogen bond to StrepTactin and biotin Site-directed mutagenesis of the key amino acids, the purpose is to retain the advantages of the original product while making up for its shortcomings, weakening the combination of biotin and StrepTactin, to obtain StrepTactin mutants (StrepTactin muts) that can reversibly bind to biotin. The 27th position was subjected to other amino acid mutations, namely S27T, S27G, S27A, S27C, S27D, S27E, S27F, S27H, S27I, S27K, S27L, S27M, S27N, S27Q, S27R, S27V, S27Y, S27P, S27W, The mutated protein sequences are respectively 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 ...

Embodiment 2

[0026] The preparation of embodiment 2, StrepTactin mut

[0027] 1) Take 5 μL of the extracted plasmid containing StrepTactin muts designed above (the plasmid containing StrepTactin mut is based on PIISA-His-StrepTactin as a template, and primers are designed for site-directed mutation. PIISA-His-StrepTactin is synthesized by the company PIISA-His-StrepTactin (as shown in SEQ ID NO.45) was added to 100 μL of BL21 codon plus (DE3) competent cells, ice-bathed for 30 minutes, then heat-shocked at 42°C for 90 seconds, then left on ice for 2 minutes, and 900 μL of LB medium was revived in a shaker at 37°C and 200 rpm for 1 hour, spread on a plate containing 100 μg / mL ampicillin resistance, and incubated overnight at a constant temperature of 37°C;

[0028] 2) On the next day, pick a single colony from the plate cultured overnight and put it into 10 mL LB medium containing 100 μg / mL ampicillin, culture it in a shaker at 37 °C and 200 rpm for 12 hours, and transfer the cultured 10 mL...

Embodiment 3

[0036] Embodiment 3, the cross-linking immobilization of StrepTactin mut

[0037] 1) Take 2mL of the purified His-StrepTactin mut protein, and dissolve the protein in 2L of 200mM NaHCO 3 , 500mMNaCl buffer solution, change the dialysate once every 2 hours, and change the dialysate twice in total;

[0038] 2) The His-StrepTactin mut protein after dialysis is measured for its ultraviolet absorption value at a wavelength of 280nm, and the protein concentration and total protein mass after dialysis are calculated according to the absorption value of 2.84 per mg of protein;

[0039] 3) Cross-link and fix 12 mg of StrepTactin mut protein per milliliter of NHS microspheres (NHS Beads), activate NHS Beads with 1 mM HCl solution 5 times the volume of Beads, and use 200 mM NaHCO with 5 times the volume of Beads after activation 3 1. Equilibrate the NHS Beads with 500mM NaCl buffer, add the dialyzed protein after the equilibrium, and rotate and cross-link at 4°C for 12h;

[0040] 4) Af...

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PUM

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Abstract

The invention discloses a mutant protein with 27th serine mutation of streptavidin and application of the mutant protein, the mutant protein is characterized in that 44-47th amino acids of wild type streptavidin are VATR, the 27th serine of the wild type streptavidin is mutated into different amino acids, the binding force between the mutated protein and Strep tag II is increased, and the mutant protein has the characteristics of high yield, high stability and the like. Compared with the prior art, the method provided by the invention has the advantages that the purification capacity on single Strep and TwinStrep fusion proteins is improved, meanwhile, the combination with Biotin is weakened, so that the action of Biotin and the combination becomes reversible, and the Biotin can be recycled after being washed and regenerated after being eluted, so that the method can be better used for purifying Biotin modified proteins and Strep tag II tag proteins.

Description

technical field [0001] The invention relates to a mutant protein with streptavidin affinity mutation, which can reversibly bind biotin after mutation, so it can be used for protein purification, regenerated and reused. Background technique [0002] Streptavidin (i.e. Streptavidin) has a super strong non-covalent binding force to 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, so it is widely used in the field of molecular biology, and its application fields include: affinity chromatography, live cell fluorescence imaging, proteomics, biotin enzyme immobilization, etc. Although streptavidin has a wide range of applications, specific to each application, there are more detailed requirements for the properties of streptavidin, such as affinity chromatography requires lower affinity and higher dissociation constant , to effectively elute target molecules ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/36C07K17/00C07K19/00C07K1/14C12N15/31C12N15/62
CPCC07K14/36C07K17/00C12N15/62C07K1/14C07K2319/22C07K2319/60C07K14/43595B01J20/24B01J20/28016C07K2319/20B01J2220/4856
Inventor 李洪涛邬文峰金瑞何红梅杨婧
Owner SOUTHWEST UNIV
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