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  • Title: Purification of branched-chain keto acid dehydrogenase regulator from Pseudomonas putida.
    Author: Madhusudhan KT, Sokatch JR.
    Journal: Methods Enzymol; 2000; 324():329-35. PubMed ID: 10989441.
    Abstract:
    BkdR can be isolated in nearly pure form as a tetramer by this procedure, which involves hyperexpressing bkdR from a plasmid, purification by chromatography on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, and dialysis to precipitate BkdR. BkdR is relatively insoluble in aqueous buffers but can be kept in solution in buffer with 50% (v/v) glycerol and 0.2 M NaCl. Cultures of E. coli DH5 alpha (pJRS119) should be maintained at 30 degrees to promote plasmid stability. Because BkdR is prone to form intermolecular disulfide bonds, buffers for SDS-PAGE should contain fresh 0.5% (v/v) 2-mercaptoethanol.
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