N dodecyl β d maltopyranoside ddm

Unless stated otherwise all chemicals used were purchased from Sigma Aldrich (Saint Louis, MO, USA); all DNA ladders, restriction enzymes and their buffers were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Primers for DNA amplification and sequencing were synthesized by Biolegio (Nijmegen, the Netherlands) or Integrated DNA technologies (Coralville, IA, USA). Sequencing services for all DNA constructs were provided by Macrogen (Amsterdam, the Netherlands). pGEM-T Easy cloning system I was purchased from Promega (Fitchburg, WI, USA). pET28a vector was purchased from Novagen (Darmstadt, Germany). EDTA-free protease inhibitor cocktail tablet was obtained from Roche diagnostics (Basel, Switzerland). n-dodecyl-β-D-maltopyranoside (DDM) was obtained from Anatrace (Maumee, OH, USA). Precision Plus Protein standards were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Isopropyl-β-D-thiogalacto- pyranoside (IPTG) was purchased from Thermo Fisher Scientific. ΔslyD BL21 (DE3) E. coli strain was a kind gift from Prof. Ry Young (Dept. Biochemistry and Biophysics, Texas A&M University, USA).

We screened multiple UlaA homologs from different prokaryotic species in order to obtain an inward-facing conformation structure. Those genes were cloned into pET21b (Novagen) with a C-terminal His x 8 tag. The transformed C43 (DE3) (Lucigen) cells were grown in Luria broth at 37 ℃ and induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) after the OD₆₀₀ reached 1.2. Cells were disrupted with a French Press with two passes at 15,000 p.s.i., in buffer A containing 25 mM Tris-Cl, pH 8.0, and 150 mM NaCl. After a low-speed centrifugation, the resulting supernatant was centrifuged at high speed to sediment a membrane fraction, which then was incubated in buffer A with 1% (w/v) n-dodecyl β-d-maltopyranoside (DDM, Anatrace) and 2 mM ascorbic acid (Sigma-Aldrich) for 1 h at 4 °C. The lysate was centrifuged again, and the supernatant was loaded onto a Ni²⁺-NTA affinity column (Qiagen). After three washes, the protein was eluted with 25 mM Tris-Cl, pH 8.0, 150 mM NaCl, 250 mM imidazole, 2 mM ascorbic acid and 0.56% (w/v) n-nonyl- β-D-maltopyranoside (NM, Anatrace), and it was then concentrated by Amicon Ultra (Millipore) for subsequent gel filtration in buffer A with detergent and 2 mM ascorbic acid. The UlaA domain from Pasteurella multocida showed a high yield and good behavior in gel filtration. The peak fractions were collected for crystallization.