Article Figures & Data
Figures
- FIG. 1.
Amino acid sequence of MazF and toxicity assay of MazF(ΔW) and MazF(ΔH). (a) Amino acid replacements in MazF(ΔW) and MazF(ΔH). In MazF(ΔH), two tryptophan residues at positions 14 and 83 in MazF were replaced with phenylalanine and leucine, respectively. In MazF(ΔH), the G27-H28 sequence was changed to KR. (b) Plate assay to compare the toxicity of pMazF with those of pMazF(ΔW) and pMazF(ΔH). Panels 1, 2, 3, 4, 5, and 6 correspond to 0.0, 0.05, 0.1, 0.25, 0.5, and 1 mM IPTG, respectively. a, b, and c correspond to pMazF(ΔW), pMazF, and pMazF(ΔH), respectively. (c) Coexpression of MazF and MazE to demonstrate that MazF toxicity was neutralized by MazE production in vivo. For induction of MazF and MazE, 0.1 mM IPTG and 0.2% arabinose, respectively, were added.
- FIG. 2.
Primer extension analysis of MazF and MazF variants. Total RNA was extracted from both the induced and uninduced cells bearing pMazF, MazF(ΔW), and MazF(ΔH). Primer extension was carried out using an ompA mRNA-specific primer as described in Materials and Methods. This result shows that identical bands were produced from all the RNAs extracted from induced cultures, suggesting their similar MazF cleavage sites (lanes 5, 7, and 9). Lanes 6, 8, and 10 represent the same from the uninduced cultures (wild type [WT]).
- FIG. 3.
Expression profiles of different proteins by using the Trp-inducible SPP system. (a) EnvZB and CspA (lanes 1 to 4 and 5 to 8, respectively); (b) E1B19K150 and GCSF (lanes 1 to 4 and 5 to 8, respectively). Lanes 1 and 5, before IPTG induction; lanes 2 and 6, after 3-h IPTG induction; lanes 3 and 7, overnight incubation in the presence of tryptophan (20 μg/ml); lanes 4 and 8, overnight in the absence of tryptophan; lanes M, molecular weight markers.
- FIG. 4.
Expression profiles of different target proteins (YaiZ, GCSF, CaM, and EnvZB) by using the His-inducible SPP system. Lanes 1, 4, 7, and 10, before IPTG induction; lanes 2, 5, 8, and 11, overnight without histidine; lanes 3, 6, 9, and 12, overnight in the presence of histidine; lane M, molecular weight markers.
- FIG. 5.
Expression of Trp-less protein for the Trp induction system. (a) pCold vectors used in this study were pColdI(SP4) (structure A), pColdI(W) (structure B), pColdII(W) (structure C), and pColdIII(W) (structure D). (b) Expression of a Trp-less human calmodulin by using the dual-induction SPP system. Lanes 1 and 4, before IPTG induction; lanes 2 and 5, after 3-h IPTG induction; lanes 3 and 6, overnight in the presence of tryptophan (20 μg/ml); lane M, molecular weight markers.
- FIG. 6.
Graphical representations of LC-MS results showing isotopic enrichment of EnvZB tryptic fragments. (a) 15N enrichment for HLFQPFVR; (b) 15N enrichment for EIETALYPGSIEVK; (c) 13C enrichment for HLFQPFVR; (d) 13C enrichment for EIETALYPGSIEVK; (e) 2H enrichment for AWFQVEDDGPGIAPEQR; (f) 2H enrichment for AVANMVVNAAR.
Tables
- TABLE 1.
Isotopic incorporation rates of several peptides generated from proteins produced by the cSPP dual-induction system
Peptide typea Isotope Peptide sequence Labeled yield (%) Labeled efficiency (%)b a 15N EIETALYPGSIEVK 97 94 HLFQPFVR 97 94 13C EIETALYPGSIEVK 97 92 HLFQPFVR 97 90 2H AWFQVEDDGPDIAPEQR 99 ND AVANMVVNAAR 99 ND b 15N FLWGSSQAK 98 82 TLDFSTPGR 99 90 13C FLWGSSQAK 98 69 TLDFSTPGR 98 89 2H FLWGSSQAK 96 67 TLDFSTPGR 99 78 15N LLLLSSVRPAIIPTEEQQ 97 89 AAAAVAFLSFIK 95 87 13C LLLLSSVRPAIIPTEEQQ 94 86 AAAAVAFLSFIK 95 88 2H LLLLSSVRPAIIPTEEQQ 94 81 AAAAVAFLSFIK 93 76 c 15N LTDEEVDEMIR 98 93 EAFSLFDKDGDGTITTK 97 88 13C LTDEEVDEMIR 97 91 EAFSLFDKDGDGTITTK 96 90 2H LTDEEVDEMIR 96 89 EAFSLFDKDGDGTITTK 93 82
