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Method for modifying protein by using transpeptidase Sortase A

A technology of transpeptidase and protein, applied in the field of genetic engineering, can solve the problems of low expression level, difficult product purification, high cost, etc.

Inactive Publication Date: 2018-01-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of Sortase A still has the following problems: (1) the expression level of Sortase A in Escherichia coli is very low; (2) the Sortase A obtained by expression needs to go through multiple steps such as cell wall breaking and separation and purification, The cumbersome process and high cost also limit the application of Sortase A; (3) Sortase A that mediates protein modification is directly added to the reaction system. After the reaction, Sortase A is not easy to separate and cannot be reused, and it will give the product purification Bring difficulty; (4) Although Sortase A being immobilized on the resin is beneficial to product purification, the reaction yield of its catalyzed protein modification is lower

Method used

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  • Method for modifying protein by using transpeptidase Sortase A
  • Method for modifying protein by using transpeptidase Sortase A
  • Method for modifying protein by using transpeptidase Sortase A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of Sortase A extracellular expression crude enzyme solution

[0022] Using pET22a as the expression vector, and using the PelB signal peptide on the vector to construct a recombinant expression vector carrying the gene encoding Sortase A (SEQ ID NO.1), the recombinant expression vector and the molecular chaperone pG-Tf2 plasmid were co-transformed into the large intestine Bacillus E. coli BL21 (DE3). With TB medium (peptone 12g / L, yeast powder 24g / L, glycerol 4g / L, KH 2 PO 4 23.1g / L, K 2 HPO 4 125.4g / L) is the fermentation medium, 50mL of liquid in 500mL shake flask, fermentation temperature 30°C, inoculum size 2%, induction time OD 600 =0.6, induction temperature 30°C, inducer IPTG concentration 100mM / L, induction fermentation time 36h. The obtained fermentation broth was centrifuged (12000rpm for 5 minutes) to remove the bacterium, and 90.2 mg / L Sortase A crude extracellular enzyme liquid could be obtained.

Embodiment 2

[0023] Example 2 Effects of Sortase A Crude Enzyme Liquid and His Tag-labeled Affinity Nickel Magnetic Beads Binding Conditions on Sortase A Enzyme Activity

[0024] The effects of the amount of Sortase A crude enzyme solution and magnetic beads, combined temperature, time and pH on the enzyme activity of magnetic beads are shown in Table 1-Table 3. It can be seen that on the basis of repeated use of magnetic beads 5 times, in order to ensure that the Sortase The catalytic effect of a common dose (10μM) requires the use of more than 3mL of Sortase A crude enzyme solution (90.2mg / L); the combined temperature, time and pH value have a significant impact on the catalytic effect of the enzyme, the pH of the crude enzyme solution When the value is 5.0-8.0, the effect of binding at 4-25°C for more than 30min is better; in addition, in order to save the cost of using magnetic beads, the enzyme activity per unit volume of magnetic beads was tested, and it was determined that the enzyme...

Embodiment 3

[0025] Example 3 Prepare a large number of Sortase A-bound magnetic beads, and detect the properties of the obtained magnetic beads

[0026]Based on the optimized binding conditions, a small amount of Sortase A-bound magnetic beads (200 μL) with activity equivalent to that of 10 μM free enzyme was successfully prepared, and on this basis, the preparation system was scaled up (10 mL). The enzymatic activity of the magnetic beads prepared by the scale-up system can reach 318.2U / 200μL magnetic beads; the microscopic examination shows that the average particle diameter increases from 19 μm to 34 μm after the magnetic beads are combined with Sortase A ( figure 1 ); As detected by SDS-Page, Sortase A in the crude enzyme solution can be completely combined after the magnetic beads are combined with Sortase A, and after elution with imidazole, it can be proved that the magnetic beads only bind the C-terminus with His 6 Labeled Sortase A( figure 2 ).

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Abstract

The invention discloses a method for modifying protein by using transpeptidase Sortase A, and belongs to the field of genetic engineering. According to the present invention, with magnetic beads capable of specifically adsorbing transpeptidase Sortase A,Sortase A is immobilized on the magnetic beads in one step so as to be used to catalyze conjugation and modification reactions of proteins or short peptides;and with the application of the prepared Sortase A conjugated magnetic beads to directly catalyze the transpeptidase conjugation reaction between the short peptide and the short peptide, the transpeptidase conjugation reaction between the protein and the Biotin, the transpeptidase conjugation reaction between the protein and the resin, the transpeptidase conjugation reaction between theshort peptide and the protein, and the like, the Sortase A conjugated magnetic beads can be recycled at least 5 times compared to the free enzyme, and can maintain the yield greater than the catalytic efficiency of free enzyme.

Description

technical field [0001] The invention relates to a method for modifying protein by using transpeptidase Sortase A, which belongs to the field of genetic engineering. Background technique [0002] Sortase A is a class of transpeptidases that mediate the covalent binding of Gram-positive bacterial cell wall-anchored proteins to the cell wall. Its application in the field of biotechnology has attracted more and more attention in recent years. Since the first report of using Sortase A as a linking tool for proteins or polypeptides in 2004, the linking reaction involving Sortase A has rapidly become a research hotspot and has been widely used in biochemistry, proteomics, biomedicine, and bioengineering technologies, etc. field. However, the application of Sortase A still has the following problems: (1) the expression level of Sortase A in Escherichia coli is very low; (2) the Sortase A obtained by expression needs to go through multiple steps such as cell wall breaking and separa...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12P21/02C12N9/52
Inventor 吴志猛赵鑫锐洪皓飞邓涛
Owner JIANGNAN UNIV
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