Figure 4.
C51/C-His expression in T. thermophilus HB27. (A) Western blot analysis. Total cellular protein was obtained by treatment of cell suspensions with trichloroacetic acid. Total protein from T. thermophilus HB27 without plasmid (lane 1) and harboring the C5 expression plasmid p16C5His (lane 2). Lane 3, prestained marker proteins with sizes (in kDa) indicated on the left. Lanes 4 and 5, the two main elution fractions from the Ni-NTA column that had been loaded with total protein derived from the plasmid-free parental HB27 strain. Lanes 6 and 7, corresponding elution fractions from the Ni-NTA column loaded with total protein derived from the HB27 strain harboring plasmid p16C5His. For details, see Materials and Methods. Asterisks mark the recombinant C51/C-His protein (encoded by plasmid p16C5His), which was further analyzed by matrix-assisted laser desorption ionization (MALDI)–time-of-flight MS (see Fig. 7). (B) Sequence of C51/C-His; black underlined letters indicate regions (positions 1–61, 89–96, and 125–171) identified by MALDI mass fingerprinting. Positions 62–88, 97–124, and 155–157 (gray letters) could not be identified. These regions are rich in Lys and Arg residues, and the tryptic fragments derived from this section of the protein would be too small to be detected by MALDI-MS under our standard conditions. The total sequence coverage is 68%.