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A protein disulfide bond isomerase modified by gene-directed modification and its application

A technology of disulfide bond isomerase and site-specific modification, applied in the field of genetic engineering, can solve problems such as low quality of bread

Active Publication Date: 2019-07-12
广州英赞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Every et al. proposed that the level of wPDI added was negatively correlated with the quality of bread, that is, the greater the amount of wPDI added, the lower the quality of bread, which may be caused by the disulfide bond reductase activity of wPDI in flour
At present, there is no relevant technical report on the application of wPDI to improve the quality of flour processing after genetic mutation

Method used

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  • A protein disulfide bond isomerase modified by gene-directed modification and its application
  • A protein disulfide bond isomerase modified by gene-directed modification and its application
  • A protein disulfide bond isomerase modified by gene-directed modification and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Transformation of wheat protein disulfide bond isomerase and its recombinant plasmid construction

[0031] The base sequence of the wild-type wheat protein disulfide bond isomerase is shown in SEQ ID NO: 1. The transformation of the wheat protein disulfide bond isomerase gene adopts overlap extension polymerase chain reaction, and the following 6 primers are designed:

[0032] R1: 5'-TTGGAGTGTCCACTCCATGGGGCGTAGAAC-3';

[0033] F2: 5'-ATGGAGTGGACACTCCAAGAGCCTGGCACC-3';

[0034] R2: 5'-TTTGAGTGTCCGCTCCAGGGTGCATAGAAC-3';

[0035] F3: 5'-TGGAGCGGACACTCAAAGAAGCTAGCACCC-3';

[0036] T7: 5'-TAATACGACTCACTATAGGG-3';

[0037] T7ter: 5'-TGCTAGTTATTGCTCAGCGG-3';

[0038] The recombinant plasmid PET30b-wPDI was used as a template, and T7 / R1, F2 / R2, and F3 / T7ter were used as primers for PCR amplification. The PCR amplification conditions were as follows:

[0039]

[0040] Three fragments were obtained after PCR amplification, and after recovery by agarose gel elect...

Embodiment 2

[0042] Example 2 Induced expression and purification of recombinant mPDI

[0043] 1. Induced expression of recombinant mPDI

[0044] Transform the constructed recombinant mPDI plasmid into BL21(DE3), pick 2 to 3 single clone colonies into LB medium containing 50 μg / mL kanamycin, and cultivate to OD at 37°C and 200r / m 600 To about 0.6-0.8, add 0.5mMIPTG to induce expression for 8h, and the induction temperature is 20°C. Through SDS-PAGE analysis, the recombinant mPDI was highly expressed ( figure 1 ).

[0045] 2. Affinity purification of recombinant mPDI

[0046] Collect the expressed bacteria, add buffer to resuspend, and use a probe-type ultrasonic instrument to disrupt the bacterial cells. The ultrasonic conditions are power 300w, duty ratio 0.4:0.6, and ultrasonic time 15 minutes. After the sonication was complete, the supernatant was collected after centrifugation at 8000r / m for 20min.

[0047] The supernatant was slowly injected into a Ni-NTA affinity chromatography co...

Embodiment 3

[0049] Example 3 Determination of Enzymatic Properties and Chaperone Activity of Recombinant mPDI

[0050] The enzymatic activity of PDI includes disulfide bond oxidase activity, reductase activity and isomerase activity. The chaperone activity of PDI refers to promoting the folding of nascent proteins and inhibiting the retrogradation of denatured proteins due to dilution. In this paper, the reductase activity of disulfide bond was characterized by insulin reduction method; the activity of disulfide bond oxidase was characterized by ribonuclease activity of recovering disulfide bond breaking; Constructase activity; Chaperone activity was characterized by inhibition of insulin B chain aggregation.

[0051] Results: As shown in Table 1, compared with wPDI, recombinant mPDI did not exhibit disulfide bond oxidase, reductase and isomerase activities, that is, lost enzymatic activity. For chaperone activity, mPDI inhibited insulin B chain aggregation to a greater extent than wPDI...

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Abstract

The invention discloses a gene fixed point reconstructed wheat protein disulfide bond isomerase and a use thereof and belongs to the field of gene engineering. Through gene fixed point reconstruction of a wild-type wheat protein disulfide bond isomerase, a wheat protein disulfide bond isomerase mutant only with chaperone activity is obtained and has an amino acid sequence shown in the formula of SEQ ID NO: 4. The amino acid sequence is obtained through fixed point reconstruction of a wild-type wheat protein disulfide bond isomerase with an amino acid sequence shown in the formula of SEQ ID NO: 3 and amino acids at 43th, 46th, 387th and 390th sites of the amino acid sequence shown in the formula of SEQ ID NO: 4 are mutated into serine (Ser) from cysteine (Cys). A bread baking result shows that the wheat protein disulfide bond isomerase subjected to gene fixed point reconstruction has bread quality improvement effects better than those of the wild-type wheat protein disulfide bond isomerase. The research result lays a foundation of deep reveal of a mechanism of wPDI chaperone activity action on gluten.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a wheat protein disulfide bond isomerase modified by gene fixed point and its application in improving the processing quality of flour. Background technique [0002] Protein disulfide isomerase (PDI) is a sulfhydryl / disulfide bond oxidoreductase located in the endoplasmic reticulum of eukaryotic cells. and important role in cell function. PDI is mainly composed of four thioredoxin domains, arranged in the order of a, b, b' and a'. Among them, a and a' contain independent CXXC active sites that catalyze thiol / disulfide bond exchange (C is cysteine ​​cys, X is an amino acid other than cys). PDI contains a variety of enzymatic activities, including disulfide bond oxidase, reductase and isomerase activities. [0003] In addition to enzymatic activity, PDI also has molecular chaperone activity, that is, it can help the nascent peptide chain to fold correctly and inhib...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21A23L7/10A23L29/00C12R1/19
CPCC12N9/90C12Y503/04001
Inventor 胡松青刘光侯轶李琳张亚萍王敬敬
Owner 广州英赞生物科技有限公司