Table 1.
Purification profiles of the heterologously produced histidine-tagged recombinant acrylyl-CoA reductases from Rhodobacter sphaeroides 2.4.1, Ruegeria pomeroyi DSS-3, and Escherichia coli K-12 substrain MG1655a
| Organism (gene locus) | Purification step | Vol (ml) | Total activity (μmol min−1) | Protein (mg) | Sp act (μmol min−1 mg−1) | Yield (%) | Purification (n-fold) |
|---|---|---|---|---|---|---|---|
| Rhodobacter sphaeroides 2.4.1 (acuI; RSP_1434) | Cell extract | 11 | 2,700 | 230 | 12 | 100 | 1 |
| Ni+ affinity chromatography | 12 | 620 | 11 | 56 | 23 | 5 | |
| Superose 12 gel filtration | 1.0 | 2.6 | 0.02 | 130 | NA | 11 | |
| Ruegeria pomeroyi DSS-3 (SPO_1914) | Cell extract | 12 | 7,100 | 310 | 23 | 100 | 1 |
| Ni+ affinity chromatography | 8.1 | 3,200 | 46 | 69 | 45 | 3 | |
| Superose 12 gel filtration | 1.5 | 32 | 0.33 | 98 | NA | 4 | |
| Escherichia coli K-12 substrain MG1655 (yhdH) | Cell extract | 12 | 3,200 | 260 | 12 | 100 | 1 |
| Ni+ affinity chromatography | 13 | 2,000 | 38 | 52 | 63 | 4 | |
| Superose 12 gel filtration | 2.0 | 14 | 0.20 | 72 | NA | 6 |
a
NA, not applicable, as only an aliquot was loaded onto the gel filtration column. In all cases, 15% (final concentration) glycerol was added to the cell extract. To the Superose 12 gel filtration column, 0.2 ml of the recombinant enzyme purified from the Ni+ affinity chromatography was applied.