Expression of C. difficile AcsB in E. coli, purification, and antibody preparation.

The C. difficile acsB gene sequence was codon optimized for expression in E. coli. The synthetic acsB gene was cloned as an untagged construct into the NcoI- and BamHI-digested pETDuet-1 plasmid (Novagen) and used for transformation of E. coli DH5α. Following sequence verification, anaerobic expression was carried out in E. coli NM522(λDE3). Anaerobic growth of E. coli and induction with isopropyl-β-d-thiogalactopyranoside (IPTG) was carried out as described previously (124). Soluble cell extracts were prepared, and the protein was purified under anaerobic conditions by anion-exchange chromatography on Q Sepharose fast flow (124). Ni reconstitution, which is required to form active acetyl-CoA synthase produced in E. coli, was modified to limit the amount of excess NiCl₂ to 2:1 (Ni/protein) and carried out at a lower pH of 7.2. Although reconstitution of other heterologously expressed AcsB proteins from archaea (Methanosarcina thermophila) and bacteria (Carboxydothermus hydrogenoformans) was effective at higher ratios of Ni/protein and higher pH, the activity of the C. difficile protein was suboptimal under those protocols. The turnover number for C. difficile AcsB in the synthesis of acetyl-CoA from CO, CoA, and methylcobinamide was 20.6 min⁻¹, assayed as described in reference 123, which compares favorably with the other AcsB proteins studied.

For immunization, the protein was further purified by extraction from SDS slab gel strips. Bands were revealed as cloudy zones of potassium dodecyl sulfate precipitate by briefly soaking the gel in buffer containing 0.25 M KCl, and strips were then cut, macerated, and extracted overnight in an excess volume of 20 mM Tris-HCl (pH 7.8) containing 0.1% SDS and 1 mM DTT. After filtration through a 0.22-μm filter, the filtrate was concentrated by ultrafiltration, diafiltered with 20 mM Tris-HCl (pH 7.8), and samples of the frozen concentrate were used for production of rabbit antisera (Covance Immunology Services, Denver, PA). Protein A-based purification of the antisera was used to produce the final anti-AcsB antibody preparation.