蛋白质硫辛酸修饰相关酶大肠杆菌研究模型的构建

doi:10.11843/j.issn.0366-6964.2019.05.018

Abstract

Abstract:

To investigate the function of lipoic acid metabolic enzymes in vivo, an Escherichia coli (E. coli) model was established.△lplA△lipB~DH5α mutant strain was obtained with deletion of lplA and lipB genes from DH5α strain using the Red homologous recombination system. The lplA and lipB genes were cloned into the expression vector of pBAD322G and transformed to the △lplA△lipB~DH5α mutant strain to construct the complementary strains of ClplA-△lplA△lipB~DH5α and ClipB-△lplA△lipB~DH5α. The lipoate modification on the substrates in the mutant and supplemented strains were analyzed with Western blot, mass spectrometric analysis and complementation experiment. The mutant strain △lplA△lipB~DH5α was very stable in the culture and displayed the phenotype of lplA and lipB deletion. Supplemented strain ClipB-△lplA△lipB~DH5α can grow in the M9 medium with glycerol as carbon source, ClplA-△lplA△lipB~DH5α can grow when LA is added to this medium. These results indicated that the E. coli model without lipoate modification function was successfully established. It will facilitate the functional study of enzymes associated with lipoic acid modification in other microorganism.

CLC Number:

S852.612

ZHU Kemeng, JIN Jin, WANG Ning, XIN Jiuqing, LIU Henggui. Establishment of E. coli Model for the Research of Lipoate-Protein Ligases[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1073-1081.

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Establishment of E. coli Model for the Research of Lipoate-Protein Ligases

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