A molecular peptide mutant
A technology of mutants and molecules, which is applied in the direction of peptides, desipeptides, peptide preparation methods, etc., can solve the problems of affecting connection efficiency, inability to form isopeptide bonds, inactivation, etc.
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Embodiment 1
[0028] The present embodiment specifies a design method for SpyCatcher-21_A82P.
[0029] The original SpyCatcher protein crystal structure used in the embodiment is obtained from the PDB database, the PDB ID is 4mli.
[0030] The structure of SpyCatcher is imported into the calculation software Rosetta, the SpyCatcher is calculated, the surface potential is set to -21, and the mutant amino acid positions are set on the surface of the protein to obtain SpyCatcher-21. The 82-bit residue of SpyCatcher-21 was then introduced into the Pro to obtain the SpyCatcher-21_A82P mutant. as Figure 1 as shown.
[0031] ADBS and VMD are used to calculate the charge density on the surface of the protein, e.g Figure 2 、 Figure 3 As shown, it can be seen that the surface of the modified molecular peptide SpyCatcher-21 carries a large amount of charge, and the mutant SpyCatcher-21_A82P also has a large amount of charge on the surface, and the potential is the same as that of SpyCatcher-21.
Embodiment 2
[0033] The present embodiment specifies a purification method for SpyCatcher-21 and SpyCatcher-21_A82P mutants.
[0034] (1) The mutated molecular peptide was synthesized in Bioengineering (Shanghai) Co., Ltd. for whole gene synthesis, and the recombinant plasmid SC-21_A82P-pET-22b was obtained on the vector pET-22b, and the enzyme resection site was Nde I and Xho I, the host is E. coli E. coli BL21(DE3)。
[0035] (2) Will be with recombinant plasmid E. coliBL21 (DE3) was cultured in LB medium at 37 °C to OD600=0.6, 1 M of IPTG was added to the final concentration of 0.5 mM, and 20 °C was induced for 10 h.
[0036] (3) After the induction is over, 8000 rpm centrifugal bacterial fluid collects the bacterial body, and adds 3 mL of phosphoric acid buffer, shock resuspending, and sonically crush at 300 W for 10 min.
[0037] (4) Ultracentrifuge the crushing liquid at 12000 rpm, 4 °C for 10 min, take the supernatant, purify the protein in Ni-NTA resin, and set aside after dialysis in ...
Embodiment 3
[0039] The present embodiment tests the connection efficiency of SpyCatcher-21 at different pHs.
[0040] According to the final concentration of 10 μM Of SpyCatcher-21 and 30 μM of SpyTag-GFP mixed, respectively, pH = 4, 5, 6, 7, 8, 9 buffer (0.1 M) in a 25 °C environment for 180 min, the connection efficiency was measured by SDS-PAGE.
[0041] Wherein the method of protein purification:
[0042] Drain 20% of the ethanol-protected liquid in the 1 mL Ni-NTA pre-loaded column and add 3-4 column volumes of BufferA to replace the ethanol in the filler. The ultracentrifuged protein sample is poured into the filler and drained. Add 3-4 column volumes of BufferA for elution, removing the heteropsy proteins adsorbed on the filler. Add another 3-4 column volume of BufferB to elute the protein of interest.
[0043] BufferA is a phosphate buffer with a pH of 8.0 0.1 M, dissolved with 500 mM of NaCl and 20 mM of imidazole;
[0044] BufferB is a phosphate buffer with a pH of 8.0 0.1 M dissolv...
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