Expression of Bovine Lactoferrin C-lobe in Rhodococcus erythropolis and Its Purification and Characterization

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  • KIM Woan-Sub
    Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University
  • SHIMAZAKI Kei-ichi
    Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University
  • TAMURA Tomohiro
    Molecular Environmental Microbiology Laboratory, Graduate School of Agriculture, Hokkaido University Proteolysis and Protein Turnover Research Group, Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST)

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  • Expression of Bovine Lactoferrin C-lobe in<i>Rhodococcus erythropolis</i>and Its Purification and Characterization

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Abstract

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 °C with shaking, and expression of the rBLF C-lobe was induced by adding 1 μg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.

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