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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, limitation of protein size and complexity, protein inactivation, etc., to avoid purification steps.

Inactive Publication Date: 2021-09-07
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 on the size and complexity of the displayed protein. In addition, target mislocalization and mutual interference between the expression of the target protein and the carrier protein may lead to protein inactivation or even degradation.
Fluorase has a complex six-subunit structure, and traditional surface display methods may cause loss of enzyme catalytic activity

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

[0036] Embodiment 1: Construction of SC-ST surface display system

[0037] 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, a strictly controlled rhaB rhamnose-inducible promoter was selected, and RBScalculator was used to design and modify the RBS on the expression vector pSQ036 (the RBS sequence is shown in SEQ ID NO.1). For the reported genes flA, SpyCatcher, SpyTag, and Lpp-OmpA, gene synthesis was carried out (see Table 1 for the nucleotide sequence), and they were 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, TriggerFactor (gene number WP_001198386.1), Lpp-OmpA, and SpyCatcher genes were connected to the expression vector pSQ036 to con...

Embodiment 2

[0042] Example 2: Heterologous Expression of Fluorase in E. coli

[0043] The recombinant plasmids pSQ036-TF-LP-SC and pSQ051 constructed in Example 1 (plasmid map such as image 3 shown) co-transformed into E.coliBL21 (DE3) to obtain the self-assembled SCST system strain; the pSQ036-LP-flA plasmid (see the plasmid map Figure 4 ) into E.coliBL21 (DE3) to obtain a traditional gene fusion surface display strain.

[0044] After plate activation, positive transformants were inoculated into LB medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate, and cultured on a shaker at 37°C for 12-16 hours. Draw 200uL and inoculate into liquid LB medium. Cultivate to OD at 220RPM, 37°C 600 When the absorbance value reaches 0.6-0.8, add IPTG with a final concentration of 0.5 mM and rhamnose with a final concentration of 10 mM, and culture overnight (about 22 hours) at 20° C. on a shaker.

Embodiment 3

[0045] Example 3: Identification of the surface display effect of fluorinase

[0046] (1) Preliminary verification of protein display on the cell surface by immunofluorescence staining

[0047] The specific implementation process is as follows:

[0048] 1. Measure the OD value of the bacterial strain cultivated in Example 2, 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;

[0049] 2. Centrifuge 1mL of the bacterial solution, resuspend it with PBS, take the calculated volume of the bacterial solution from the upper part, add the corresponding volume of 4% PFA (paraformaldehyde, freshly prepared) to form a 1mL system, and react for about 15 minutes;

[0050] 3. Centrifuge the reacted bacterial solution to remove PFA, add 1mL 1%-3% BSA (Bovine Serum Albumin bovine serum albumin, ready-to-use) to resuspend, and seal for 30min;

[0051] 4. Take 20uL of the reaction solution to incubate wi...

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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 and the 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 fluorinase 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 restricted. 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 biom...

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
CPCC12N15/70C12N9/1085C12Y205/01063C12N2830/002C07K2319/03C07K2319/00
Inventor 周景文杨文涵余世琴堵国成陈坚
Owner JIANGNAN UNIV
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