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QSOX, mutant protein thereof, as well as preparation and use

A mutant and protein technology, applied in the field of QSOX and its mutant protein and preparation, can solve the problem of undiscovered yeast

Inactive Publication Date: 2009-08-12
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

QSOX proteins are present in all multicellular organisms with known genomes but not in yeast

Method used

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  • QSOX, mutant protein thereof, as well as preparation and use
  • QSOX, mutant protein thereof, as well as preparation and use
  • QSOX, mutant protein thereof, as well as preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Construction and expression of DPICZaA-Q6604 vector system

[0099] Total RNA was extracted from fibroblasts using Trizol reagent. The extracted total RNA was used as a template, reverse-transcribed into cDNA by RT-PCR, using primers P1: 5'-GTActc gag aaa agaATGAGGAGGTGCAACAGCGGCTCCG---3' and P2: 5'-CAGgcg gcc gcTCAAATAAGCTCAGGTCCCTCAG-3', amplified by PCR The QSOX gene coding sequence was obtained, and the QSOX gene fragment was loaded into the pGEM-T vector. Then it was digested with Xhol I and Not I, and subcloned into pPICZaA to obtain pPICZaA-Q6604. Sequencing results and analysis such as Figure 2A and Figure 2B shown.

[0100] After the correctly constructed pPICZaA-Q6604 was linearized with SalI, the yeast was electrotransformed according to the method in "Molecular Cloning: Experiment Guide". The obtained recombinant yeast was screened for copy number by Zeocin resistance, identified correctly by PCR, and induced expression with methanol as ...

Embodiment 2

[0101] Example 2 Construction and expression of pET28a-Q6604-70m vector system

[0102] See figure 1As shown, using the QSOX gene loaded on the pGEM-T vector as a template, using primers Pm70-1-5'GGAGTTCTTCGCCTCCTGGGCCGGCCACGCCATCGCCTTCGCCC-3 and Pm70-2-5GGGCGAAGGCGATGGCGTGGCCGGCCCAGGAGGCGAAGAACTCC-3 to mutate the Cys at positions 70 and 73 of the QSOX protein to Ala, and obtain The mutated gene was double digested with NdeI and Not I, and then directional cloned into pET28a. After the obtained recombinant vector was identified correctly, it was transformed into Escherichia coli, and the recombinant strain was induced in LB liquid medium with a final concentration of 0.3mM IPTG at 18°C ​​for 24 hours for small expression, and QSOX enzyme binding factor FAD10μM was added during the induction, and the induction ended At 6000rmp, the supernatant was centrifuged to collect the precipitate. Recombinant protein QSOX was expressed in Escherichia coli to obtain soluble protein....

Embodiment 3

[0103] Example 3 Construction and expression of pTYB12-Q6575 vector system

[0104] Using the QSOX gene loaded into the pGEM-T vector as a template, PCR amplification was performed using primers P30N5'tatcatatgGCCCCGCGGTCGGCGCTCTATTCGCCTT-3' and P2: 5'-CAGgcg gcc gcTCAAATAAGCTCAGGTCCCTCAG-3', and the amplified product was QSOX without the signal peptide The coding sequence of the gene is called Q6575, and the QSOX gene fragment is double digested with NdeI and Not I, and then directionally cloned into pTYB12. After the obtained recombinant vector was identified correctly, it was transformed into Escherichia coli, and the recombinant strain was induced in LB liquid medium with a final concentration of 0.3mM IPTG at 18°C ​​for 24 hours for small expression, and QSOX enzyme binding factor FAD10μM was added during the induction, and the induction ended At 6000rmp, the supernatant was centrifuged to collect the precipitate. Recombinant protein QSOX was expressed in Escherichia...

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Abstract

The invention obtains a full length coding region cDNA sequence of a QSOX protein through an RT-PCR method. Then fixed point mutation is carried out; wild type genes and mutant genes are respectively constructed into pET series, pGEX series and pTYB series of vectors of pronucleus expression vectors and yeast expression series vectors so as to convert Escherichia coli and yeast for inductive expression and carry out the purification and function study on an expressed product.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to QSOX and its mutant protein, as well as its preparation method and application. Background technique [0002] Disulfide bond formation is an important protein post-translational modification, which determines whether the protein can fold correctly and finally form an active spatial conformation. With the in-depth research on a series of enzymes related to protein folding, such as disulfide isomerase (PDI), sulfhydryl oxidase (QSOX, ERV / ALR family), people have a new understanding of disulfide bonds. Many current research results have shown that disulfide bonds in proteins are regulated, and its formation and reduction can change the biological activities of proteins and enzymes, and then have a major impact on cell metabolism regulation, signal transduction and other functions. [0003] The QSOX family is the newly discovered sulfhydryl oxidase family. It utilizes a thioredoxin dom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N15/70
Inventor 杨弋郑文云尹琴徐磊
Owner EAST CHINA UNIV OF SCI & TECH
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