FIG. 1.
Immunoblots of C-terminally His-tagged Amt proteins in cell extracts. All proteins were tagged with six histidine residues. Cell extracts were subjected to SDS-10% (A and B) or 12% (C) polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blotted with a specific anti-His antibody (INDIA HisProbe-HRP; Pierce). Plasmids for panels A and C were derived from vector pET21a (Novagen). The host strain was BL21(DE3)(pLysE). Plasmids for the experiment in panel B were derived from pJES1335. The host strain was NCM2019 (10). Expression of Amt proteins was induced as described in the text for purification of A. aeolicus AmtB. (A) Lane 1, E. coli AmtB (pJES1139); lane 2, A. aeolicus AmtB (pJES1331); lane 3, M. jannaschii Amt1 (pJES1345); lane 4, M. jannaschii Amt2 (pJES1346). (B) Lane 1, A. aeolicus AmtBT253I, T259M, S404F (pJES1378); lane 2, A. aeolicus AmtB (pJES1335). (C) Lane 1, A. aeolicus AmtBΔ2-51 (pJES1432); lane 2, AmtBΔ2-87 (pJES1433): lane 3, AmtBΔ2-143 (pJES1434); lane 4, AmtBΔ2-170 (pJES1435); lane 5, AmtBΔ2-209 (pJES1436); lane 6, AmtBΔ398-423 (pJES1437); lane 7, molecular weight standards; lane 8, A. aeolicus AmtB (pJES1331).