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Crude extract of BL21 DE3 (pREP4GroESL) transformed with 349 pET2818 after IPTG induction (15 µg of protein); lane 2, crude extract of BL21 DE3 350 (pREP4GroESL) transformed with pML118 after IPTG induction (15 µg); lane 3, protein fraction 351 eluting from metal affinity chromatography with 100 mM imidazole (5 µg); lane 4, protein 352 fraction eluted with 100-200 mM NaCl from the anion-exchange column (4 µg) ,
The major muropeptide 360 monomers generated by mutanolysin (peak 2) and AtlA (peak 2') were analyzed by MS/MS 361 yielding fragmentation patterns (A) and (B), respectively. The m/z values of the most informative 362 ions are boxed and the inferred structures are indicated with a one-letter code: M, MurNAc; M R , 363 reduced MurNAc, p.364 ,
10 µg, lane 1) was digested with proteinase K and aliquots were 372 withdrawn after 1 min (lane 2), 5 min (lane 3) or 10 min (lane 4). The three polypeptides A, B C 373 (indicated by an arrow) were subjected to N-terminal sequencing. (D) Zymogram showing cell 374 ,
Proteins were separated by SDS-PAGE in a gel containing 0, pp.2-375 ,