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Purification of branched-chain keto acid dehydrogenase regulator fromPseudomonas putida

Elsevier Science & Technology
DOI: 10.1016/s0076-6879(00)24242-0
  • Section Ii. Cloning
  • Expression
  • And Purification Of Enzymes Of Branched-Chain Amino Acid Metabolism


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% (vv) glycerol and 0.2 M NaCl. Cultures of E. coli DH5α (pJRS119) should be maintained at 30° to promote plasmid stability. Because BUR is prone to form intermolecular disulfide bonds, buffers for SDS-PAGE should contain fresh 0.5% (vv) 2-mercaptoethanol.

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