IdeS (immunoglubulin G-degrading enzyme of streptococcus pyogenes) protease and preparation method and application thereof
A protease and fragment technology, applied in the field of biological enzymes, can solve the problems of difficult large-scale application and low expression level, and achieve the effects of high expression level, uniform product, and high specificity
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Embodiment 1
[0038] Embodiment 1: Preparation of IdeS protease—Escherichia coli
[0039] The nucleotide sequence of the IdeS protease is obtained by gene synthesis technology, and the nucleotide sequence of the IdeS protease is recombined into the expression vector pET32a to construct a recombinant expression vector. The recombinant expression vector proved correct by the sequencing is transformed into the expression bacteria BL21 Escherichia coli to obtain the positive expression bacteria containing the IdeS protein gene sequence.
[0040] Specifically, 6 single colonies were picked and cultured overnight at 37°C. Add the overnight cultured bacterial solution to 5mL Amp+LB medium at a ratio of 1:100, and incubate at 220rpm at 37°C for 2-3h. Take out 1 mL of the bacterial liquid as the uninduced control, and add IPTG to the rest according to the volume to a final concentration of 0.5 mM, and incubate at 37 ° C for 4 h at 180 rpm. Take 100uL of the bacterial solution and centrifuge at 120...
Embodiment 2
[0042] Embodiment 2: Preparation of IdeS protease—Pichia pastoris
[0043] The nucleotide sequence of the IdeS protease is obtained by gene synthesis technology, and the nucleotide sequence of the IdeS protease is cloned into an expression vector pPICα to construct a recombinant expression vector. The recombinant expression vector proved to be correct by the sequencing is transformed into KM71H yeast by electroporation to obtain a positive expression bacterium containing the IdeS protein gene sequence.
[0044] Specifically, 10 single colonies were picked and inoculated into a 125ml shake flask containing 10ml of BMGY medium. Culture at 30°C, 250rpm for 2 days. All the cells were collected and centrifuged, and the cells obtained by centrifugation were resuspended with 1 mL of BMMY medium, and cultivated overnight at 30°C and 300 rpm. On the second day, 100 μL of the bacterial liquid was taken, and 100 μL of 40% methanol was added for induction, and cultured at 30° C. and 300...
Embodiment 3
[0045] Embodiment 3: the industrialized production of IdeS protease
[0046] The Ides protease is obtained by Escherichia coli fermentation culture, and gradually expanded to 1000L fermentation tank scale fermentation production through shake flask culture. Escherichia coli grows in carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements, and antifoaming agents can be added to control foam, such as antifoaming agent Antifoam204.
[0047] It is produced by fed-batch feeding and submerged aerobic culture, and glucose or glycerol is selected as the limiting carbon source to control fed-batch feeding. The fermentation temperature is 37°C, and pH 7.0 is controlled by adding 25% ammonia water; the ventilation rate of the fermenter is selected as 0.5-2.0vvm, and the dissolved oxygen value is about 30% by controlling the feed rate, stirring rate and ventilation rate. During the fermentation and cultivation process, samples were taken regularly to measure the op...
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