Expression and purification method of totipotent endonuclease

A technology of expression purification and nuclease, which is applied in the fields of genetic engineering and protein engineering, can solve the problems of high price of medium raw materials, high production cost, and strictness, and achieve the effect of simple culture conditions, easy operation, and reduced difficulty

Pending Publication Date: 2019-11-19
上海药明康德新药开发有限公司
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  • Abstract
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Problems solved by technology

[0003] At present, the commercialized totipotent nuclease is secreted and expressed from recombinant Pichia pastoris. The culture conditions of

Method used

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  • Expression and purification method of totipotent endonuclease
  • Expression and purification method of totipotent endonuclease

Examples

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Embodiment 1

[0038] In this example, first, through genetic modification, a 6xHis tag was added to the front end of the totipotent nuclease gene sequence to construct a recombinant plasmid (see S1 for the expressed protein sequence), introduce it into Escherichia coli, and inoculate it in the basal medium. When OD600 reaches 0.6-0.8, add inducer IPTG with a final concentration of 0.1-1 mM, and induce expression at 30 degrees for 8 hours or 25 degrees for 16 hours.

[0039] S1: Protein expression sequence:

[0040] mgsshhhhhhdlgtenlyfq*gsMSIDNCAVGCPTGGSSKVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCK

[0041] Among them, hhhhhh: indicates the 6xHis tag; enlyfq*g: indicates the TEV restriction site, and * indicates the restriction position; capital letters indicate the totipotent nuclease sequence.

[0042] Formula ...

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Abstract

The invention discloses an expression and purification method of a totipotent nuclease. The method comprises the following steps: (1) expression steps: E. coli containing a target gene growing in a basic culture medium, adding an inducer isopropyl-P-D-thiogalactose to induce expression of a target protein, the target gene containing a totipotent nuclease sequence, the front end of the totipotent nuclease sequence containing a tag for coding 6 x His, and the target protein being an inclusion body; (2) inclusion body renaturation: diluting and renaturing the inclusion body obtained in the step (1) to obtain the soluble and active target protein; and (3) purification steps: conducting affinity chromatography for purification, enzyme digestion and inversion of the target protein to obtain thetotipotent nuclease with high purity. According to the present invention, the production cost of the totipotent endonuclease can be effectively reduced, the purification time can be shortened, and thetotipotent endonuclease with high activity can be obtained by the provided method, and the method is conducive to the application of the totipotent endonuclease in scientific research and industrialfields.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and protein engineering, and specifically relates to a method for expressing and purifying a totipotent endonuclease. Background technique [0002] The totipotent nuclease monomer contains 245 amino acid residues, the molecular weight is 30kDa, and the two monomers are connected by two disulfide bonds to form an active dimer structure. Altipotent nuclease is an endonuclease that can efficiently degrade DNA and RNA in any form (including double-stranded, single-stranded, linear, and circular) into 5' monophosphate nucleotides of 2 to 5 bases . Altipotent nuclease can effectively reduce the viscosity of protein samples and remove nucleic acid contamination in protein samples. While reducing the viscosity, it also has many other uses, such as: reducing the sample processing time, increasing the protein yield, separating the precipitate and the supernatant more thoroughly during centrif...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/70
CPCC12N9/16C12N15/70
Inventor 李颖洁刘一璇杨青
Owner 上海药明康德新药开发有限公司
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