semaglutide recombinant synthesis Total Synthesis of Semaglutide Based on a Soluble Hydrophobic-Support-Assisted Liquid-Phase Synthetic Method How Ozempic Works: (Bio)Chemical Perspectives

Embodiment 3 [0053] Expression and purification of embodiment 3 fusion protein [0054] Transfer the seed liquid of the recombinant positive engineering strain of Example 2 to LB medium according to the inoculum size of 2%, cultivate it at 37C until OD600=0.6, add 50 l of 0.5mol / L IPTG, induce at 15C for 16 hours, and collect by centrifugation For each 1 g of wet bacteria, resuspend in 10 mL of lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH 8.0, 17 l (10 mg / mL PMSF)), ultrasonicate and centrifuge to obtain supernatant and precipitate

The desired monocistronic (single copy) of His6-AV-Ubiquitin- semapeptide (nucleotide sequence)-encoding gene PCR product of ~320bp was obtained as evident by 1% (w/v) Agarose gel electrophoresis (Figure 1)

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A solution of compound of formula-3a (0.2 gm) in THF (22 ml) was added to the reaction mixture at 15-20C and stirred the reaction mixture for 4 hr at the same temperature

and (4) carrying out gradient elution by taking the reverse-phase silica gel filler as the stationary phase, an aqueous solution of ammonium bicarbonate as the mobile phase A and acetonitrile as the mobile phase B, collecting fractions, carrying out rotary evaporation, and carrying out freeze-drying so as to obtain saltless refined peptide of semaglutide, wherein the saltless refined peptide has purity of greater than 99.5% and an individual impurity content of less than 0.1%

In the method, by introducing the Fmoc-Val10-Ser11 (Psi(Me, Me) pro)-OH to solve the problem of difficult sequence synthesis of the semaglutide, difficult sequence synthesis becomes simple and easy