Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (5): 98-107.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1149
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BAI Fu-mei1(), LI Zhi-min2, WANG Xiao-qin1, HU Zi-wei1, BAO Ling-ling1, LI Zhi-min1,3()
Received:
2020-09-09
Online:
2021-05-26
Published:
2021-06-11
Contact:
LI Zhi-min
E-mail:bfm18270860135@163.com;zmlizm@126.com
BAI Fu-mei, LI Zhi-min, WANG Xiao-qin, HU Zi-wei, BAO Ling-ling, LI Zhi-min. Biochemical Characterization and Structural Analysis of N-acetylornithine Transaminase from Synechocystis sp. PCC6803[J]. Biotechnology Bulletin, 2021, 37(5): 98-107.
Primer | Primer sequence(5'-3') | Restriction enzyme |
---|---|---|
slr1022-F | GGAATTCCATATGACCTATTCC-CCTGTTGTTGAATC | Nde I |
slr1022-R | CCGCTCGAGTCAAACCAAAGT-GGCGATCGCCTGAC | Xho I |
Table 1 PCR primer sequences
Primer | Primer sequence(5'-3') | Restriction enzyme |
---|---|---|
slr1022-F | GGAATTCCATATGACCTATTCC-CCTGTTGTTGAATC | Nde I |
slr1022-R | CCGCTCGAGTCAAACCAAAGT-GGCGATCGCCTGAC | Xho I |
Fig. 1 Cloning of slr1022 gene from Synechocystis sp. PCC6803 and construction of its recombinant plasmid pET28a-slr1022 A: PCR amplification product of slr1022 gene. M: DNA marker. Lane 1-2: PCR product. B: Double enzymatic digestion products of slr1022 gene and pET-28a vector. M: DNA marker. Lane 1-2: Double enzymatic digestion product of pET-28a vector and slr1022 gene, respectively. C: Identification of recombinant plasmid pET28a-slr1022. M: DNA marker. Lane 1: Positive control. Lane 2-8: Colony PCR product
Fig. 2 Expression of recombinant Slr1022 protein M: Protein marker. Lane 1-3: Expression of pET-28a plasmid (cell lysate, supernatant and pellet, respectively). Lane 4-6: Expression of pET28a-slr1022 recombinant plasmid (cell lysate, supernatant and pellet, respectively)
Fig. 3 SDS-PAGE of recombinant Slr1022 protein A: Purification of recombinant Slr1022 protein. M: Protein marker. Lane 1-3: Cell lysate, supernatant and flow through. Lane 4-8: 20, 40, 60, 80, 100 mmol/L imidazole elution. 9-14: 200 mmol/L imidazole elution. B: The purified Slr1022 protein. M: Protein marker. Lane 1: The purified Slr1022 protein
Fig. 4 Kinetic profiles of recombinant Slr1022 protein A: Plot of the initial velocities as function of N-acetylornithine concentrations, the concentration of α-ketoglutarate was fixed at 1 mmol/L. B: Plot of the initial velocities as function of α-ketoglutarate concentrations, the concentration of N-acetylornithine was fixed at 2 mmol/L. Data are expressed as the mean ± standard deviation, n = 3
Fig. 6 Protein sequences alignment of N-acetylornithine aminotransferases from different sources slr1022, 2E54, 6W7X, 2EH6 and 2PB0 were N-acetylornithine aminotransferases from Synechocystis PCC6803, Thermotoga maritima, Stenotrophomonas maltophilia, Aquifex aeolicus and Salmonella typhimurium respectively. Residues interacting with PLP are marked by▲, and★ indicates the residues forming Schiff base with PLP and ◆ indicates the residues from the other subunit which interact with PLP
Fig. 7 Homologous modeling structure of Slr1022 protein A: Cartoon structure of Slr1022 protein. B: Enlarged schematic diagram of active site. C: The conserved amino acid residues in the active site. The colors of α-helix, β-strands and loops are depicted as red, yellow and green, respectively. The cofactor PLP and the conserved amino acid residues are shown as sticks, and their carbon atoms are light blue and green, respectively. The yellow dotted lines indicate the hydrogen bonds interacting with PLP. The modeled structure is depicted by SWISS-MODEL based on the structure of N-acetylornithine aminotransferase of Thermotoga maritima (PBD: 2E54)
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