Figure 2.
Intracellular localization, phenotype, and yield of Tat-targeted MBP. (a) Western blot analysis of total soluble lysate (Lys), cytoplasmic (Cyt), periplasmic (Per) and insoluble (Ins) fractions from HS3018 (malE-deficient derivative of MC4100) cells expressing wildtype (wt) MBP and ssTorA-MBP with 0, 6 or 10 additional residues of mature TorA (+0, +6, +10). Western blots were probed with anti-MBP primary antibody. (b) HS3018 and HS3018 ΔtatC cells expressing the above constructs streaked on MacConkey agar media supplemented with 0.4% maltose. (c) Yield of the above constructs following IMAC purification from the periplasm of HS3018 cells. Data represents the average of 3 replicate experiments. (d) Amino acid sequence of the TorA (+10) signal peptide highlighting the primary signal peptidase cleavage site (white triangle) and the second cleavage site (grey triangle) as reported by N-terminal sequencing.