, 1989) with the appropriate antibiotics. All plasmids were purified from E. coli DH5α using the Large Scale Nucleobond® check details AX plasmid isolation kit (Macherey-Nagel, Oensingen, Switzerland). The yahD gene was PCR-amplified from genomic DNA of L. lactis IL1403, using the following primers: sm10 (5′-TGAATGGAGGAGGACATATGACAGATTATGTT; NdeI-site underlined) and sm11 (5′-TGCACTGAAACTTTTTCAATAC). The PCR product was inserted into the TOPO-TA cloning vector® (Invitrogen life Technologies), resulting in pTHB. The yahD gene was excised from pTHB with NdeI and NotI and ligated into the pTYB12 expression vector (IMPACT™-CN system, New England Biolabs), cut with the

same enzymes, resulting in pSMI. Escherichia coli Rosetta (Novagen, UK) transformed with pSMI was grown to the mid-log phase at 37 °C in LB broth containing 100 μg mL−1 ampicillin, cooled to 18 °C and induced with 1 mM isopropyl-β-d-thiogalactopyranoside. Following overnight growth, cells were harvested by centrifugation at 3000 g for 10 min at 4 °C. Cell pellets were resuspended in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, pH 8.0, and frozen at −20 °C. To the thawed HKI-272 nmr cell suspension, a tablet of EDTA-free complete protease inhibitor cocktail (Roche, Switzerland) and 10 μg mL−1 of DNaseI (Roche) was added and cells were

disrupted by three pressure cycles in an Emulsiflex C5 homogenizer (Avestin, Germany) at 1000–1500 bar at 4 °C. Debris was removed by centrifugation at 4 °C for 10 min at 3000 g. The supernatant was centrifuged at 90 000 g for 30 min at 4 °C, filtered and YahD was absorbed to chitin beads (New

England Biolabs) in the batch mode for 1 h at 4 °C. Intein cleavage was induced with 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA and 50 mM dithiothreitol, pH 8.0, on a column. YahD-containing fractions eluted from the chitin column were concentrated on an Amicon-Ultra-10k centrifugal filter (Millipore) and further purified by size exclusion chromatography on a Superdex S-75 26/60 column (GE Healthcare, Germany) in 10 mM Tris-Cl, 150 mM NaCl and 1 mM EDTA, pH 8.0. Fractions were analyzed by electrophoresis on 12% sodium dodecylsulfate Bumetanide (SDS) polyacrylamide gels and stained with Coomassie blue as described (Laemmli & Favre, 1973). Fractions containing pure YahD were pooled and concentrated as before to 10 mg mL−1. Protein concentrations were determined with the BioRad protein assay (BioRad, Richmond) using bovine serum albumin as a standard. The total masses of purified, native YahD as well as YahD incubated overnight with 0.1 mM phenylmethylsulfonylfluoride were analyzed by matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF-MS) using an Ultraflex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a Smart beam™ laser. Sinapinic acid was used as a matrix.