High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose‐binding protein as a solubility tag

Abstract

Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to Δ(1) -pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

“…3B). This indicates non-cooperative unfolding for the MBP and TtProDH domains46. The first transition reflects unfolding of MBP, with a midpoint of unfolding around 55 °C.…”

“…Cell lysate was centrifuged at 26000 g for 1 h at 4 °C. Apo-EE was purified using an amylose column (New England Biolabs, 20 mL in XK 16/10), and a Source 15Q column (GE Healthcare, 20 mL in XK 16/10), according to a protocol that has been described before for the holoenzyme4647. In addition, after the ion exchange column, the enzyme was concentrated using a 10 kDa cut off Amicon filter and loaded on a preparative Superdex200 XK26/1000 column (GE Healthcare), equilibrated in 50 mM sodium phosphate, 150 mM NaCl, pH 7.4.…”

“…Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Thermo Scientific) and the purified apoenzyme with a concentration of 7.5 mg/mL was flash-frozen in liquid nitrogen and stored at −80 °C. The native mass of apo-EE was determined using nanoflow electrospray ionisation mass spectrometry (ESI-MS), as described before4647.…”

“…MBP-TtProDH ΔABC was produced and purified as described previously47. The denatured mass of MBP-TtProDH ΔABC was determined using nanoflow electrospray ionisation mass spectrometry (ESI-MS)46. Removal of the MBP-tag and purification of native ΔABC was achieved by slight modification of the trypsin-treatment reported before46.…”

“…Previously, we described the properties of Thermus thermophilus ProDH (TtProDH), produced through fusion with maltose-binding protein (MBP)46. Because MBP-TtProDH appeared to be prone to aggregation, we constructed a variant with a more polar N-terminus (F10E/L12E).…”