Oligonucleotide primers were synthesized by Integrated DNA Technologies or Sigma-Aldrich. Recombinant plasmid DNA was purified with a QIAprep® Spin Miniprep Kit from Qiagen. Gel extraction of DNA fragments and restriction endonuclease clean up were performed using an Illustra GFX PCR DNA and Gel Band Purification Kit from GE Healthcare. DNA sequencing was performed by Beckman Coulter Genomics and Eton Bioscience. Optical densities of E. coli cultures were determined with a DU 730 Life Sciences UV/Vis spectrophotometer (Beckman Coulter) by measuring absorbance at 600 nm. All chemicals and solvents were obtained from Sigma-Aldrich except where noted.
During protein purification, proteolysis was inhibited by adding Pierce™ Protease Inhibitor tablets (EDTA free; Thermo Scientific) to lysis buffer. Affinity chromatography and SDS-PAGE analysis were conducted using HisPur™ Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (Thermo) and 10% or 4–15% Mini-PROTEAN TGX Precast SDS-PAGE gels (Bio-Rad) with 2× Laemmli sample buffer (Bio-Rad). Protein concentrations were determined by measuring absorbance at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo).
During protein purification, proteolysis was inhibited by adding Pierce™ Protease Inhibitor tablets (EDTA free; Thermo Scientific) to lysis buffer. Affinity chromatography and SDS-PAGE analysis were conducted using HisPur™ Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (Thermo) and 10% or 4–15% Mini-PROTEAN TGX Precast SDS-PAGE gels (Bio-Rad) with 2× Laemmli sample buffer (Bio-Rad). Protein concentrations were determined by measuring absorbance at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo).