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Method for surface display of fluorinase based on self-assembly

A surface display and self-assembly technology, applied in the field of genetics and bioengineering, can solve the problems of loss of enzyme catalytic activity, lack of surface display technology, protein inactivation, etc., and achieve the effect of avoiding purification steps

Inactive Publication Date: 2021-03-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional gene fusion method has limitations such as limitations in the size and complexity of the displayed protein, target mislocalization, and mutual interference between the expression of the target protein and the carrier protein, resulting in protein inactivation or even degradation. Fluorase has a complex six-subunit structure. Surface display methods may result in loss of enzyme catalytic activity
[0004] Therefore, the lack of a new surface display technology limits the surface display of fluorinase, making it impossible to apply microbial methods to industrial production.

Method used

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  • Method for surface display of fluorinase based on self-assembly
  • Method for surface display of fluorinase based on self-assembly
  • Method for surface display of fluorinase based on self-assembly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Construction of SC-ST surface display system

[0033] Using the plasmid co-expression system, two plasmids with different replicons and resistances were selected to express the target protein. For the anchoring protein Lpp-ompA, the strictly controlled rhaB rhamnose-inducible promoter was selected, and the RBS was designed and modified using RBScalculator. The reported genes flA, SpyCatcher, SpyTag, and Lpp-OmpA were chemically synthesized, respectively connected to T vectors for preservation. Primers were designed to amplify the target gene from the T vector, and the PCR product was purified after verification by DNA agarose gel to obtain the cloned target gene. Using the Gibson assembly method, Trigger Factor (gene number WP_001198386.1), Lpp-OmpA, and SpyCatcher gene were connected to the expression vector to construct plasmid pSQ036-TF-LP-SC ( figure 2 ); Lpp-OmpA forms a complex protein expression with SpyCatcher, and Trigger Factor is independentl...

Embodiment 2

[0039] Example 2: Heterologous Expression of Fluorase in E. coli

[0040] The recombinant plasmid pSQ036-TF-LP-SC constructed in Example 1 was transferred into E.coliBL21(DE3), and the recombinant bacteria E.coliBL21(DE3) / SC was obtained through verification, and then the recombinant bacteria E.coliBL21(DE3) ) / SC to make a competent state, transfer pSQ051 into E.coliBL21(DE3) / SC-ST, and obtain positive transformants after sequencing verification, which are recombinant bacteria.

[0041]Insert the constructed recombinant bacteria into LB medium, add kanamycin and ampicillin at a final concentration of 50 μg / L, and culture at 37°C; Add to LB medium and cultivate at 37°C; the cell concentration reaches OD 600 Take it out when the temperature is 0.8-1, put it in a shaker at 20° C. to cool down for 10-15 minutes, add an inducer to continue culturing overnight (the inducer is 0.5 mM IPTG and 15 mM rhamnose at a final concentration).

Embodiment 3

[0042] Example 3: Identification of the surface display effect of fluorinase

[0043] (1) Preliminary verification of protein display on the cell surface by immunofluorescence staining

[0044] The specific implementation process is as follows:

[0045] 1. After culturing, measure the OD value of the bacteria and calculate the final OD 600 is 1, the dilution factor and the volume of the bacterial solution required for the final volume to be 1 mL;

[0046] 2. Centrifuge 1 mL of the bacterial solution, resuspend it with PBS, take the volume of the calculated bacterial solution from the upper part, add the corresponding volume of 4% PFA (paraformaldehyde, freshly prepared) to form a 1 mL system, and react for about 15 minutes.

[0047] 3. Centrifuge the reacted bacterial solution to remove PFA, add 1 mL of 1%-3% BSA (Bovine Serum Albumin bovine serum albumin, ready-to-use) to resuspend, and block for 30 minutes.

[0048] 4. Take 20uL of the reaction solution for incubation wit...

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Abstract

The invention discloses a method for displaying fluorinase on the surface based on self-assembly, and belongs to the field of genes and bioengineering. Through a modular method, the target protein andthe carrier protein are independently expressed, and surface display of the complex protein can be realized through in-situ assembly after translation. A SC-ST system is used in the invention, so that the technical scheme realizes the display of the hexasubunit structure fluorinase on the surface of Escherichia coli, avoids complex purification steps, can improve the downstream separation and purification efficiency, and saves the cost. The application of the fluorinating enzyme in industrial production is favorably realized.

Description

technical field [0001] The invention relates to a method for displaying fluorinase on a surface based on self-assembly, which belongs to the field of gene and bioengineering. Background technique [0002] Fluorine (F, ninth in the element cycle), ranks 24th in the ratio of naturally occurring elements among all elements on the earth, and ranks the highest in the ratio of naturally occurring halogen elements in the earth's crust, but it is relatively rare in the form of soluble fluorine ions . Fluorine is easily solvated in aqueous solution, which greatly reduces its biological activity. Compared with other halogens, it is difficult to generate high value-added organic compounds through haloperoxidase oxidation. This makes the bioavailability of fluorine extremely limited. Although naturally occurring fluorinated organics are rare, the value of fluoride is enormous. Organic fluorides can be widely used in the fields of bioorganic chemistry, medicinal chemistry, and biomate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N1/21C12N9/10C12R1/19
CPCC07K2319/00C07K2319/03C12N9/1085C12N15/70C12N2830/002C12Y205/01063
Inventor 周景文杨文涵余世琴堵国成陈坚
Owner JIANGNAN UNIV
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