The extracellular secretion of EGF-LARDs in E. coli XL1-Blue and MM294. A. The secretion of EGF-LARDs in E. coli XL1-Blue. Each culture harboring one of pEGF-LARD1-4 (lanes 1–4) or pEGF-TliA (lane A) with pEcPrtDEF was centrifuged twice, and the supernatant was collected. The growth conditions were identical to those in Figure 2. SDS-PAGE with 15% gel and western blotting with anti-LARD antibodies were performed on the supernatant. The (+) sign indicates the presence of PrtDEF. The (−) sign indicates the absence of PrtDEF (pACYC-184 was used as a negative control). Purified TliA at 99 μg/ml concentration was used as an index after dilution. B. The secretion of EGF-LARDs in E. coli MM294. The methods used here were identical to those used in (A).