Gene Cloning, Site-Directed Mutation, Protein Expression and Purification

The RdacuH gene was amplified from the genome of Roseovarius nubinhibens ISM by PCR using FastPfu DNA polymerase. PCR primers were designed with the NdeI and XhoI restriction sites. The amplified RdacuH is recombined to the vector pET-22b. FastPfu DNA polymerase and the vector pET-22b were purchased from TransGen Biotech (China) and Novagen company (Germany), respectively.

All site-directed mutations were introduced using overlap PCR and verified by sequencing. The constructed recombinant plasmids, including the wild type RdacuH and its mutants were transferred into E. coli BL21 (DE3) for expression. The cells were cultured in LB medium with 0.1 mg/ml ampicillin at 37°C to an OD₆₀₀ of 1.0-1.2. Then the culture was induced at 20°C overnight with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Wild type RdAcuH and its mutants were purified by affinity chromatography on a Ni²⁺-NTA column (GE healthcare, United States), and then fractionated by anion exchange chromatography on a Source 15Q column (GE healthcare, United States) and gel filtration on a Superdex G200 column (GE healthcare, United States).

The prpE gene was amplified from the genomic DNA of Dinoroseobacter shibae DFL 12. Expression and purification of PrpE were conducted with the same methods as RdAcuH described above.