Recombinant Escherichia coli and application thereof to fermentative production of 2[4Fe4S]ferredoxin

A technology of recombinant Escherichia coli and Escherichia coli, applied in the directions of fermentation, microorganism-based methods, bacteria, etc., can solve the problems of high protein price, low expression amount, low activity, etc., and achieve good application prospects, high expression amount, and cost saving Effect

Inactive Publication Date: 2016-01-27
INST OF BASIC MEDICINE OF SAMS
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

At present, only the [2Fe2S] type ferredoxin from spinach is commercially sold. However, due to species differences, this ferredoxin shows different efficiencies when analyzing enzymes from other species, and the protein is very expensive , which largely limits the application of ferredoxin and scientists' research on ferredoxin-related enzymes and metabolic processes
[0003] In the existing literature reports, most ferredoxins are purified from wild bacteria and other organisms, and are also prepared by heterologous expression, but these methods have many shortcomings, such as consuming a lot of time and cumbersome purification steps , low expression, incomplete iron-sulfur cluster assembly of ferredoxin and low activity, low final yield and purity, etc.
Based on this, the research and development of a method capable of efficiently expressing 2[4Fe4S]ferredoxin has become an urgent problem to be solved. After searching, the recombinant Escherichia coli capable of efficiently expressing 2[4Fe4S]ferredoxin and its production in fermentation The application of 2[4Fe4S]ferredoxin has not been reported

Method used

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  • Recombinant Escherichia coli and application thereof to fermentative production of 2[4Fe4S]ferredoxin
  • Recombinant Escherichia coli and application thereof to fermentative production of 2[4Fe4S]ferredoxin
  • Recombinant Escherichia coli and application thereof to fermentative production of 2[4Fe4S]ferredoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of recombinant Escherichia coli that highly expresses 2[4Fe4S]-type ferredoxin in C.pasteurianumDSM525

[0035] Take C.pasteurianumDSM525 cells, and extract genomic DNA from C.pasteurianumDSM525 cells according to the method recommended by the bacterial genomic DNA extraction kit. According to the known 2[4Fe4S] type ferredoxin gene sequence in the genome of Clostridium pasteurianum (C.pasteurianum) DSM525 as shown in SEQ ID NO.1, the primers were designed as follows:

[0036] F Primer: 5'GACGACGACAAGATGGCATATAAAATCGCTGATTCATGTG3'

[0037] R primer: 5'GAGGAGAAGCCCGGTTATTCTTGTACTGGTGCTCCAACTGGA3'

[0038] Using the extracted C. pasteurianum DSM525 genomic DNA as a template, KOD hot-start DNA polymerase was used for PCR amplification to obtain 2[4Fe4S]-type ferredoxin gene. The PCR reaction system is as follows:

[0039]

[0040] The PCR reaction conditions are:

[0041] 95℃5min

[0042] 95°C for 30s, 55°C for 30s, 72°C for 30s and 30 cycles ...

Embodiment 2

[0048] Embodiment 2: the application of recombinant escherichia coli highly expressed 2 [4Fe4S] ferredoxin

[0049] (1) Inoculate the recombinant Escherichia coli strain constructed in Example 1 into the TB medium containing antibiotics and iron-sulfur sources with an inoculum size of 1% by volume, and cultivate it at 37°C and 180rpm for 2 to 3 hours to OD 620nm is 0.6, the bacterial liquid is obtained;

[0050] Among them, the formula and final concentration of components of the above TB medium are: Tryptone12g / L, Yeastextract24g / L, Glycerol4ml / L, KH 2 PO 4 2.3g / L, K 2 HPO 4 12.5g / L;

[0051] The above-mentioned antibiotics are chloramphenicol with a final concentration of 25 μg / ml, tetracycline with a final concentration of 10 μg / ml, and carbenicillin with a final concentration of 25 μg / ml;

[0052] The formula and final concentration of the above-mentioned iron and sulfur sources are: ferric ammonium citrate 0.2-0.3g / L, L-cysteine ​​0.1-0.2g / L, ferric citrate 0.18-0.3g...

Embodiment 3

[0056] Example 3: Separation, purification and activity determination of 2[4Fe4S]ferredoxin.

[0057] The purification of protein was carried out in an anaerobic operation box, and all solutions used were degassed and anaerobic treated after boiling.

[0058] Prepare the buffer required for purification: buffer A: Tris-HCl100mM, NaCl150mM, DTT2mM, pH7.5; buffer B: sodium phosphate Tris-HCl100mM, NaCl150mM, DTT2mM, desthiobiotin2.5mM, pH7.5; buffer W: Sodium phosphate 50mM, pH7.0.

[0059] Acquisition of crude enzyme solution: resuspend the frozen cells prepared in Example 2 with buffer A containing 10% glycerol, and use an ultrasonic disruptor to disrupt the cells. The cell disruption procedure is: working time 6 seconds, intermittent time 6 seconds , power 39%, total crushing time is 50 minutes. After the crushing is completed, centrifuge at 12,000 rpm for 30 minutes, take the supernatant and filter it with a 0.22um pore size filter membrane, and place it on ice as a crude ...

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Abstract

The invention discloses a recombinant Escherichia coli. The strain contains a 2[4Fe4S]ferredoxin gene with a nucleotide sequence as shown in SEQ ID NO.1, and is named as Escherichia coli pCodonPlus/pRKISC/pET51b-2[4Fe4S]Fd which is obtained by the steps of connecting the 2[4Fe4S]ferredoxin gene into a pET51b carrier to prepare a recombinant plasmid named as pET51b-2[4Fe4S], and transferring the prepared recombinant plasmid to E.coli C41(DE3) containing pCodonPlus and pRKISC plasmids. The invention also discloses application of the recombinant Escherichia coli to fermentative production of 2[4Fe4S]ferredoxin. Experiments prove that each litre of fermented cultures can be used for generating 3.5mg of purified 2[4Fe4S]ferredoxin, and the yield is far higher than 0.5mg of 2[4Fe4S]ferredoxin purified from wild bacteria, and therefore, the recombinant Escherichia coli disclosed by the invention has a favorable application prospect in industrial production.

Description

technical field [0001] The invention relates to gene recombination engineering bacteria and its application, in particular to a recombinant Escherichia coli containing 2[4Fe4S]ferredoxin gene and its application in fermentative production of 2[4Fe4S]ferredoxin. Background technique [0002] Ferredoxin is a kind of acidic protein containing iron-sulfur clusters, and the iron-sulfur clusters are mainly divided into [4Fe4S], [2Fe2S], [3Fe4S] and other types. As a common electron carrier, ferredoxin participates in important metabolic processes such as respiration, photosynthesis, and fermentation. At present, only the [2Fe2S] type ferredoxin from spinach is sold commercially. However, due to species differences, this ferredoxin shows different efficiencies when analyzing enzymes from other species, and the protein is very expensive , which largely limits the application of ferredoxin and scientists' research on ferredoxin-related enzymes and metabolic processes. [0003] In t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/02C12R1/19
Inventor 黄海燕王书宁胡烈杰许晓群王郡甫栾俊文苏庆红
Owner INST OF BASIC MEDICINE OF SAMS
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