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Method for producing recombinant high-activity manganese superoxide mutase

A superoxide and dismutase technology, applied in the biological field, can solve problems such as low activity and incorrect folding process

Inactive Publication Date: 2011-01-26
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the problems in the production of genetically engineered Mn-SOD is that because the overexpression after induction is too late to fold correctly to form insoluble inclusion bodies, further denaturation and renaturation are required to obtain active Mn-SOD. In addition, even soluble expression, and generally have the problem of low activity, which is also caused by the incorrect folding process

Method used

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  • Method for producing recombinant high-activity manganese superoxide mutase
  • Method for producing recombinant high-activity manganese superoxide mutase
  • Method for producing recombinant high-activity manganese superoxide mutase

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1, the soluble expression of recombinant human Mn-SOD

[0054] Design the following primers:

[0055] Forward: 5-GACATATGAAGCACAGCCTCCCCGACC-3' (SEQ ID NO: 1);

[0056] Reverse: 5'-GCAAGCTTGCATAACGATCGTGGTTTAC-3' (SEQ ID NO: 2).

[0057] Using human placenta cDNA (purchased from Invitrogen) as a template, PCR amplification was performed with the aforementioned primers to obtain the coding sequence of human Mn-SOD.

[0058] The sequence obtained above was digested with NdeI / HindIII and inserted into the corresponding site of the pET28a expression vector (purchased from Invitrogen), and sequenced to identify the correctly inserted recombinant expression vector. The recombinant expression vector was transformed into Escherichia coli BL21(DE3) by conventional methods to obtain transformants.

[0059] Pick the monoclonal colony on the transformation plate, inoculate it into 30mL LB culture medium (containing 100μg / mL Amp), place it on a shaker at 37°C for overn...

Embodiment 2

[0060] Embodiment 2, adding different concentrations of Mn in the supernatant 2+ Effect on Mn-SOD activation

[0061] The cell culture obtained above was ultrasonically disrupted, and the supernatant was collected after centrifugation, which contained soluble recombinant human Mn-SOD and other miscellaneous proteins.

[0062] Add different concentrations of Mn to the supernatant containing soluble recombinant human Mn-SOD (rhMn-SOD) 2+ , observe different concentrations of Mn 2+ Effect on Mn-SOD activation. The results are shown in Table 1.

[0063] The determination method of rhMn-SOD activity adopts the trace pyrogallol method. Enzyme activity is defined as follows: at 25° C., the amount of enzyme that inhibits the autoxidation rate of pyrogallol to 50% per minute in 1 ml of reaction solution is an activity unit.

[0064] The determination of rhMn-SOD specific activity is defined as: the number of enzyme activity units contained in each mg protein. The unit is U / mg.

...

Embodiment 3

[0068] Embodiment 3, thermal denaturation method removes impurity protein in supernatant

[0069] The cell culture obtained above was ultrasonically disrupted, and the supernatant was collected after centrifugation to obtain a crude enzyme solution.

[0070] The crude enzyme solution obtained after ultrasonically crushing the bacteria was subjected to heat denaturation for 5, 10, 15, and 20 minutes in a water bath at 60, 65, 70, 75, and 80°C, respectively, and immediately immersed in water (water temperature 0°C) to cool after heat denaturation, and centrifuged After 10 min, take the supernatant enzyme solution and measure the specific activity. The obtained specific activity assay results are shown in figure 1 a.

[0071] It can be seen that the thermal denaturation effect at 75°C for 10 minutes is the best, and the specific activity in the supernatant is increased by 2.5 times, and the total enzyme activity is not significantly reduced at this time, see figure 1 b.

[00...

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Abstract

The invention relates to a method for producing recombinant high-activity manganese superoxide mutase. In the method, soluble expression manganese superoxide mutase is recombined, cell supernatant is subjected to thermal denaturation, protein solution is purified, and the product of the purification is activated by manganese ions at a proper concentration. The method can produce high-activity manganese superoxide mutase.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a method for producing recombinant highly active manganese superoxide dismutase. Background technique [0002] Superoxide dismutase (SOD) is a metalloenzyme widely present in organisms, which can protect aerobic cells from the poisoning of reactive oxygen species (ROS) produced during normal metabolism. Catalyzes the following reactions: 2O 2 .- +2H + →H 2 o 2 +O 2 , H 2 o 2 It is subsequently cleared by catalases (CATs) and peroxidases. [0003] There are a variety of isoenzymes in the SOD family, which can be divided into copper-zinc superoxide dismutase (Cu, Zn-SOD) and manganese superoxide dismutase (Mn-SOD) according to the different metal prosthetic groups contained in the molecule. , iron superoxide dismutase (Fe-SOD) and extracellular superoxide dismutase (EC-SOD), these four types of SOD all catalyze the same catalytic reaction. Because d...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12R1/19
Inventor 李素霞袁勤生陈车生吴梧桐
Owner EAST CHINA UNIV OF SCI & TECH
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