An engineering bacterium producing human selenoprotein p mutant and its application
A protein and product technology, applied in the field of engineering bacteria producing human selenoprotein P mutants, can solve the problems of difficult recombinant expression of human selenoprotein P, cumbersome purification steps, and low yield
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Embodiment 1
[0048] Embodiment 1, the synthesis of SePm gene
[0049]According to Genbank's human selenoprotein P gene sequence (NM_005410 sequence), the selenocysteine codon TGA was mutated into cysteine codon TGT (or TGC), combined with the codon preference characteristics of Escherichia coli genes (codon usage Frequency), the original human selenoprotein P gene SeP was changed to the human selenoprotein P mutant gene SePm. The human selenoprotein P mutant gene SePm was obtained by whole gene synthesis.
[0050] 1. Artificially synthesize the following primers:
[0051] R0:5'-CACCACCCACCCATATGC-3'
[0052] F01: 5'-G CATATG GGTGGTGGTGGTACTGAATCTCAAGATCAGAGC-3'
[0053] R17: 5'-CGGTTGTTTGCACAGAGAGCTCTGATCTTGAGATTCAGTAC-3'
[0054] F40:5'-TCTCTGTGCAAACAACCGCCAGCATGGAGCATTCG-3'
[0055] R58:5'-GCATCGGATCTTGGTCACGAATGCTCCATGCTGG-3'
[0056] F75:5'-TGACCAAGATCCGATGCTGAACTCCAATGGTTCCG-3'
[0057] R92:5'-GCAGTGCAACGACAGTTACGGAACCATTGGAGTTCA-3'
[0058] F110:5'-TAACTGTCGTTGCACTGCTGC...
Embodiment 2
[0129] Embodiment 2, the construction of the engineering bacterium that produces human selenoprotein P mutant
[0130] 1. The SePm gene synthesized in Example 1 was connected to the pUCm-T vector to obtain a recombinant plasmid, which was named TSePm, and the TSePm was sequenced, and the result was correct.
[0131] 2. Digest TSePm with NdeI and XhoI to obtain the gene fragment; use NdeI and XhoI to double-digest pET-30a (+) to obtain a large fragment of the vector; connect the gene fragment to the large fragment of the vector to obtain the recombinant plasmid pET-SePm, The pET-SePm was sequenced and the result was correct.
[0132] 3. Transform pET-SePm into Escherichia coli JM109 (DE3) by chemical method, screen positive transformants with LB plates containing 50ug / ml kanamycin sulfate, and obtain a strain producing human selenoprotein P mutant SePm, which Named ZH2Y-SePm, the strain was stored in a 15% glycerol tube at -80°C for future use.
[0133] Transform pET-30a (+) ...
Embodiment 3
[0134] Embodiment 3, identification of human selenoprotein P mutant SePm protein
[0135] 1. Inoculate the ZH2Y-SePm strain into LB medium containing 50ug / ml kanamycin sulfate, and culture it on a shaker at 37°C and 200rpm for 12h.
[0136] 2. Add IPTG to the bacterial solution to a final concentration of 1 mM, and after inducing expression for 5 hours, take the bacterial solution out of the shaker, collect bacteria by centrifugation, and obtain bacterial precipitates.
[0137] 3. After adding the bacterial lysate to the bacterial pellet, ultrasonically disrupt the bacterial cells, and then centrifuge to obtain the supernatant.
[0138] 4. Purify the supernatant from step 3 through a nickel column (HiTrapChelating prepacked column). The eluted target protein was identified by SDS-PAGE protein electrophoresis, and the results were as follows figure 1 shown.
[0139] figure 1 Among them, 1 is protein molecular weight standard; 2 is the component purified by nickel column.
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