High-expression semeglutide precursor recombinant engineering bacterium and construction method thereof
A technology of recombinant engineering bacteria and semaglutide, which is applied in the field of genetic engineering, can solve the problem of low fermentation expression and achieve high industrial application value and prospects
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Embodiment 1
[0036] Example 1: Construction of recombinant Escherichia coli engineering bacteria that stably and highly express semaglutide precursor
[0037] (1) Synthesize the fusion polypeptide encoding genes shown in SEQ ID No.7 and SEQ ID No.8 respectively, and add Nde I enzyme cutting site, 3' end add stop codon and xho I restriction site;
[0038] (2) The gene fragments prepared in step (1) were respectively cloned into the expression vector pET-30a(+) by enzyme digestion. Nde I and xho Between the I enzyme cutting sites, thereby constructing two kinds of recombinant expression vectors pET-30a(+)-A1-GLP-1 and pET-30a(+)-A2-GLP-1;
[0039] (3) Transform the recombinant expression vectors obtained in step (2) into Escherichia coli BL21(DE3) by heat shock method, and select 8 clones after resistance screening, and select 8 clones from the two recombinant engineering bacteria, and name them respectively A1-GLP-1-1 to A1-GLP-1-8 and A2-GLP-1-1 to A2-GLP-1-8 were stored in glycerol....
Embodiment 2
[0041] Example 2: Fermentation of recombinant Escherichia coli engineering bacteria that stably and highly express semaglutide precursor
[0042] (1) Recovery of recombinant engineered bacteria
[0043] Take each of the 16 strains of glycerol bacteria preserved in Example 1, and insert them into 16 flasks containing 20 ml of sterilized LB medium according to the inoculation amount of 1 / 1000 respectively, and put them under the conditions of 37°C and 200rpm Cultivate overnight to obtain a recovery bacterial solution.
[0044] (2) Induced expression of recombinant engineered bacteria
[0045] Take the resuscitated bacteria solution in step (1) and put them into 16 flasks containing 50 ml of sterilized LB medium according to the inoculation amount of 1%, and cultivate them to OD at 37°C and 220rpm 600 When the value reaches 0.6-0.8, add IPTG with a final concentration of 0.5mM, and induce expression overnight at 37°C and 220rpm.
[0046] (3) Cell collection and HPLC expression...
Embodiment 3
[0050] Example 3: High-density fermentation of recombinant Escherichia coli engineering bacteria with stable and high expression of semaglutide precursor
[0051] Get the 16 strains of recombinant engineered bacteria constructed in Example 1 and carry out the secondary bacterial classification activation respectively, carry out high-density fermentation, in the fermentation process, after feeding starts, every 2h sampling measures OD 600 value, and draw the growth curve; after the fermentation, the cells were collected by centrifugation.
[0052] According to the fermentation results, A1-GLP-1 series recombinant engineering bacteria and A2-GLP-1 series recombinant engineering bacteria can both ferment to high OD 600 value, especially the A2-GLP-1 series of recombinant engineering bacteria, can reach OD 600 When the value is above 240, the biomass of inclusion bodies reaches above 300g / L. Taking A2-GLP-1-5 as an example, its fermentation OD 600 The value can reach 259, and t...
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