Expression of bovine lactoferrin C-lobe in Rhodococcus erythropolis and its purification and characterization.
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 degrees C with shaking, and expression of the rBLF C-lobe was induced by adding 1 microg/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.
Kim
Woan-Sub
WS
Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan. wwsskim@hotmail.com
Shimazaki
Kei-ichi
K
Tamura
Tomohiro
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004926:Escherichia coli / Q000187:drug effects; D015870:Gene Expression; D007781:Lactoferrin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D007781:Lactoferrin