A kind of extraction method of insulin glargine precursor protein
A technology for insulin glargine and its extraction method, which is applied in the field of extraction of insulin glargine precursor protein, can solve the problems that the recovery rate of the precursor protein is not significantly improved, the protein content is not increased, and the content of the insulin glargine precursor protein is increased , Make up for the loss of the target protein and improve the production efficiency
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Embodiment 1
[0028] Microbial strain: Pichia pastoris GS115 (commercially available)
[0029] C peptide sequence: amino acid sequence shown in SEQ ID NO:1
[0030] Fermentation broth: obtained by fermentation culture of Pichia pastoris GS115 introduced with the precursor gene of insulin glargine.
[0031] The pH of the fermentation broth was measured to be 5.0, then the supernatant was centrifuged and the concentration of the precursor of insulin glargine was measured by HPLC as a standard value of 100 as a control, and then 16 parts of the fermentation broth of the same volume were taken, using 1.2mol / L hydrochloric acid and glacial acetic acid , 10% concentrated sulfuric acid and 95% phosphoric acid were adjusted to pH 1, 2, 3, 4, and then centrifuged to take the supernatant for comparison. The results are shown in Table 1.
[0032] Table 1 HPLC detection results of insulin glargine precursors adjusted by different acids
[0033] pH1234 5 (control) 1.2mol / L hydrochloric acid173179171129100 ...
Embodiment 2
[0036] Microbial strain: Pichia pastoris GS115 (commercially available)
[0037] C peptide sequence: amino acid sequence shown in SEQ ID NO:1
[0038] Fermentation broth: same as Example 1
[0039] The pH value of the fermentation broth was measured to be 5.0, and then the supernatant was centrifuged and the concentration of the precursor of insulin glargine was measured by HPLC as a standard value of 100 as a control. Then 10 parts of the fermentation broth of the same volume were taken, using 1.2mol / L hydrochloric acid and 95% Phosphoric acid was adjusted to pH 1.2, 1.5, 1.8, 2.1, 2.4, and then centrifuged to take the supernatant for comparison. The results are shown in Table 2.
[0040] Table 2 HPLC detection results of insulin glargine precursors adjusted by different acids
[0041] pH1.21.51.82.12.4 5 (control) 1.2mol / L hydrochloric acid170172172179165100 95% phosphoric acid133137160185178100
[0042] It can be seen from Table 2 that adding acid treatment according to the met...
Embodiment 3
[0044] Microbial strain: Saccharomyces cerevisiae MT663 (commercially available)
[0045] C peptide sequence: amino acid sequence shown in SEQ ID NO:1
[0046] Fermentation broth: obtained by fermentation culture of Saccharomyces cerevisiae MT663 introduced with the precursor gene of insulin glargine.
[0047] The pH of the fermentation broth was measured to be 5.2, then the supernatant was centrifuged and the concentration of the precursor of insulin glargine was measured by HPLC as a standard value of 100 as a control, and then 10 parts of the fermentation broth of the same volume were taken, using 1.2mol / L hydrochloric acid and 95% Phosphoric acid was adjusted to pH 1.2, 1.5, 1.8, 2.1, 2.4, and then centrifuged to take the supernatant for comparison. The results are shown in Table 3.
[0048] Table 3 HPLC detection results of insulin glargine precursors adjusted by different acids
[0049] pH12345 5.2 (Control) 1.2mol / L hydrochloric acid161170160119107100 95% phosphoric acid13...
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