Method for improving expression of selenoprotein TrxR

A technology of selenoproteins and culturing strains, which is applied in the field of improving the expression of selenoprotein TrxR, can solve the problems affecting the separation and purification of selenoproteins, restricting the research of selenoproteins, and the low expression of selenoproteins. The method is simple, the raw materials are easy to obtain, and the cost is low. cheap effect

Active Publication Date: 2018-09-18
DALIAN UNIV OF TECH
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  • Abstract
  • Description
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Problems solved by technology

[0004] The existing selenoprotein expression methods generally have the problem of low expression level, which greatly affects the subsequent separation and purification of selenoproteins and limits further research on selenoproteins

Method used

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  • Method for improving expression of selenoprotein TrxR

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Embodiment 1

[0024] Use 250ml glass Erlenmeyer shaker flask experiment, add 50ml LB medium, 50μl 15mg / ml tetracycline, 50μl 50mg / ml kanamycin and 50μl 34mg / ml chloramphenicol, 1ml inoculum. Use a shaker to culture at 37°C and 220rpm until the end of logarithmic growth, add 50μl of 0.5M IPTG, 50μl of 5mM selenite, 50μl of 100mg / ml L-cysteine ​​and 2.5-15μl of 1M MnCl 2 , and then low temperature induced expression at 24°C for 24h. Take out 1ml of the bacterial solution, centrifuge to discard the supernatant, add 50μl TE buffer, 1μl 1mg / ml lysozyme, freeze and thaw 3 times repeatedly to dissolve the broken bacterial protein, centrifuge at 13,000rpm for 10min, collect the supernatant as the crude enzyme containing TrxR liquid. Use the DTNB method to test the activity, take the enzyme volume activity as the index, and find that Mn 2+ The expression of TrxR can be promoted in a certain concentration range. When Mn 2+ When the concentration is 100μM, the effect of promoting expression is bet...

Embodiment 2

[0026] Use 250ml glass Erlenmeyer shaker flask experiment, add 50ml LB medium, 50μl 15mg / ml tetracycline, 50μl 50mg / ml kanamycin and 50μl 34mg / ml chloramphenicol, 1ml inoculum. Use a shaker to culture at 37°C and 220rpm until the end of logarithmic growth, add 50μl of 0.5M IPTG, 50μl of 5mM selenite, 50μl of 100mg / ml L-cysteine ​​and 5-50μl of 1M CaCl 2 , and then low temperature induced expression at 24°C for 24h. Take out 1ml of the bacterial solution, centrifuge to discard the supernatant, add 50μl TE buffer, 1μl 1mg / ml lysozyme, freeze and thaw 3 times repeatedly to dissolve the broken bacterial protein, centrifuge at 13,000rpm for 10min, collect the supernatant as the crude enzyme containing TrxR liquid. Use the DTNB method to test the activity, take the enzyme volume activity as the index, and find that Ca 2+ The expression of TrxR can be promoted in a certain concentration range. When Ca 2+ When the concentration is 400μM, the effect of promoting expression is bette...

Embodiment 3

[0028] Use 250ml glass Erlenmeyer shaker flask experiment, add 50ml LB medium, 50μl 15mg / ml tetracycline, 50μl 50mg / ml kanamycin and 50μl 34mg / ml chloramphenicol, 1ml inoculum. Use a shaker to culture at 37°C and 220rpm until the end of logarithmic growth, add 50μl of 0.5M IPTG, 50μl of 5mM selenite, 50μl of 100mg / ml L-cysteine ​​and 5-25μl of 1M MgCl 2 , and then low temperature induced expression at 24°C for 24h. Take out 1ml of the bacterial solution, centrifuge to discard the supernatant, add 50μl TE buffer, 1μl 1mg / ml lysozyme, freeze and thaw 3 times repeatedly to dissolve the broken bacterial protein, centrifuge at 13,000rpm for 10min, collect the supernatant as the crude enzyme containing TrxR liquid. Use the DTNB method to test the activity, take the enzyme volume activity as the index, and find that Mg 2+ The expression of TrxR can be promoted in a certain concentration range. When Mg 2+ When the concentration is 300μM, the effect of promoting expression is bette...

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Abstract

A method for improving expression of selenoprotein TrxR is disclosed and belongs to the technical field of protein prokaryotic expression. In a prokaryotic expression process of the TrxR, an expression strain is first cultured under conditions of 37 DEG C and 220 rpm until the end of the logarithmic phase, then L-cysteine, sodium selenite, isopropyl thiogalactopyranoside (IPTG), and divalent metalions such as Mn<2+>, Ca<2+>, and Mg<2+> are added to the medium, and inducible expression under conditions of 24 DEG C and 220 rpm is performed for 24 h. The addition of the divalent metal ions suchas Mn<2+>, Ca<2+>, and Mg<2+> enhances the expression of the TrxR. The method is simple, raw materials are easily available, the cost is low, expression of the TrxR is obviously improved, and the method has high utilization value.

Description

technical field [0001] The invention belongs to the technical field of protein prokaryotic expression and provides a method for improving the expression of selenoprotein TrxR. Background technique [0002] Mammalian thioredoxin reductase (thioredoxin reductase, TrxR) is a very important class of selenoproteins. TrxR regulates cell proliferation, activity and apoptosis by regulating the activity and redox state of thioredoxin (Trx), which is closely related to the pathogenesis of tumors and other diseases, and maintains the redox balance of organisms. Among them, TrxR is the most widely distributed in the cytoplasm and nucleus. [0003] For the expression of selenoproteins, Rengby et al. obtained the "2.4 / 24 / 24" expression method of selenoprotein biosynthesis, added the inducer IPTG at the end of the logarithm (OD600nm=2.4), and harvested the cells after induction of expression at 24°C for 24 hours. Regulation of selenoprotein expression (Rengby O, Johansson L, Carlson L A,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
CPCC12N9/0051C12Y108/01009
Inventor 许建强张竞争徐卫平孙世博关水马昆薛宏宇王晓晓张楠
Owner DALIAN UNIV OF TECH
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