FIG. 3.

Effect of amplification of the ileX, argU, and leuW genes in E. coli BL21(λDE3) on production of light chains of BoNT/A (A), BoNT/B (B), BoNT/C (C), and BoNT/E (D). The expression of each protein was evaluated in the presence or absence of amplified ileX, argU, or leuW genes, as follows: lane 1, no amplification (pACYC184); lane 2, pACYC-Ile7; lane 3, pACYC-Arg34; lane 4, pACYC-L10; lane 5, pACYC-IleArg10; lane 6, pACYC-RL5; and lane 7, pACYC-IRL10. Cells were induced with IPTG and lysed, and the total cell proteins were separated by 4 to 20% gradient SDS–PAGE and visualized by Coomassie blue staining. The arrows indicate the locations of the various neurotoxin proteins. The molecular weight markers used (lane M) were phosphorylase b (molecular weight, 97,400), bovine serum albumin (66,200), glutamate dehydrogenate (55,000), ovalbumin (42,700), aldolase (40,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme (14,400).