The intracellular expression of EGF-LARDs in E. coli XL1-Blue. Each E. coli XL1-Blue culture harboring one of pEGF-LARD1-4 (lanes 1–4) or pEGF-TliA (lane A) with pEcPrtDEF (+) or pACYC-184 (−) was incubated with 0.05 mM IPTG, 50 μg/ml ampicillin, and 34 μg/ml chloramphenicol in 2 ml glass tubes at 37°C. The cells were isolated from the extracellular medium by double centrifugation, underwent SDS-PAGE in 15% gel, and analyzed by western blotting using anti-LARD antibodies. The plasmid pACYC-184 was used as a negative control for pEcPrtDEF which encodes PrtDEF. Purified TliA at a concentration of 99 μg/ml was used as an index after dilution. The arrowheads indicate the size of each EGF-LARD.