Skip to main content
. 1998 Jul 21;95(15):8491–8495. doi: 10.1073/pnas.95.15.8491

Figure 1.

Figure 1

Construction of plasmids for overexpression of fusion proteins. Two vectors (from New England Biolabs) were used for constructing the fusions. plH1148 contains the malE gene terminated by the coding sequences for His-Tyr (the amino acid sequence required for the genenase protease). The malE gene in this vector is deleted for the signal sequence so that the fusion protein is expressed in the cytoplasm. pMAL-p2 contains malE terminated by the coding sequence for Ile-Glu-Gly-Arg (required for the protease factor Xa) and contains the signal sequence so that the fusion protein is secreted into the periplasm. Two overexpression vectors were used for the constructs: pSRT7, which contains ptsI, the gene encoding EI, under control of the inducible T7 polymerase (pT7 promoter), and pET21a, which also contains pT7. pET21a was converted to pET∷MAL by inserting malE, which had been severed from pIH1148 with NdeI and HindIII. A DNA fragment encoding EI-C was cut from pSRT7 with BglII and HindIII, and the desired gene fusions were constructed by inserting the fragment downstream of the malE genes in pMAL-p2 and pET∷MAL, giving the overexpression vectors pMAL∷EI-C/Xa and pMAL∷EI-C/G, respectively.