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. 2002 Aug;184(15):4054–4064. doi: 10.1128/JB.184.15.4054-4064.2002

FIG. 3.

FIG. 3.

Separation of proteins from cell extracts of R. eutropha JMP134, JMP222, and derivatives by means of a MonoQ HR5/5 anion-exchange (A to C) or a phenyl-Superose HR5/5 hydrophobic-interaction column (D to F). Cell extracts were either directly applied (A to C) or applied after addition of ammonium sulfate (final concentration of 1 M) (D to E), and proteins were eluted with a linear gradient of NaCl (0 to 0.5 M) or (NH4)2SO4 (1 to 0 M). The eluted fractions (0.5 ml) were analyzed for activities against the respective assay substrates (usually applied at a concentration of 50 μM, except for catechol and 3-chlorocatechol, which were added at a concentration of 200 μM). Enzymes eluting with a yield of >40% are shown. Arrows, enzymes eluting with a low yield (>20%; TfDFI and TfdFII). Elution was performed in Tris-HCl buffer, except for panel C, where phosphate buffer was used. (A) Separation of a cell extract of 3CB-grown cells of JMP222(pBBR1M-I) (6.1 mg of protein). (Inset) Activity of chromosomally encoded maleylacetate reductase after separation of a cell extract of 4-fluorobenzoate (4FB)-grown cells of JMP222 (7.9 mg of protein). (B) Separation of a cell extract of 3CB-grown cells of JMP222(pBBR1M-II) (11.6 mg of protein). (Inset) Detailed view of the activities observed in fractions 30 to 41. (C) Separation of a cell extract of 3CB-grown cells of JMP222(pBBR1M-I) (5.6 mg of protein). (Inset) Activity of chromosomally encoded maleylacetate reductase after separation of a cell extract of 4FB-grown cells of JMP222 (6.5 mg of protein). Muconate cycloisomerase activities could not be recovered after elution with phosphate buffer, as phosphate completely inhibited those enzymes. (D) Separation of a cell extract of 3CB-grown cells of JMP222(pBBR1M-I) (4.7 mg of protein). (Inset) Activity of chromosomally encoded maleylacetate reductase after separation of a cell extract of 4FB-grown cells of JMP222 (8.1 mg of protein). (E) Separation of a cell extract of 3CB-grown cells of JMP222(pBBR1M-II) (4.5 mg of protein). (F) Separation of a cell extract of 2,4-D-grown cells of JMP222(pBBR1M-I) (3.6 mg of protein).