Fig. 3
From: Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4
Bacterial growth and hIL-10 production over time. a Culturing recombinant BGN4 graph at OD600nm. b Quantification of hIL-10 produced by BGN4 [pBESIL10] Cell Free Culture Supernatant (CFCS) during culture in pH-controlled MRS medium by sandwich ELISA. (c) Western blot detection of human IL-10 protein. a The 120 mL of cultured recombinant bacteria were centrifuged and inoculated into 1.2 L of a fresh MRS broth (pH 6.5) at 0 h each. BGN4 cells were cultured until reaching a pH of 6.0, and a diluted ammonia solution was added to maintain a pH of 6.8 during the bacteria culture. b The pH was maintained at 6.8 during cultivation and the negative control BGN4 [pBES2] CFCS did not show detectable hIL-10 at the same condition. All data are shown as the mean ± standard deviation (n = 4). The amount of hIL-10 were: (2 h) 128 ± 46 ng/mL, (3 h) 189 ± 54 ng/mL, (4 h) 327 ± 20 ng/mL, (5 h) 554 ± 42 ng/mL, (6 h) 956 ± 129 ng/mL, (7 h) 1,124 ± 170 ng/mL, (8 h) 1,171 ± 114 ng/mL, (9 h) 1,416 ± 300 ng/mL and (10 h) 1,473 ± 300 ng/mL, respectively. (c) All CFCS were concentrated 40-fold by different precipitation methods. Lanes 1, 3, 5: BGN4 [pBES2] control strain grown in buffered MRS medium (pH 6.8); Lanes 2, 4, 6, 7: BGN4 [pBESIL10] strain grown in buffered MRS medium (pH 6.8); Lane C: positive control (recombinant hIL-10 protein (1 μg)); Lanes 1, 2: ethanol precipitation; Lanes 3, 4: acetone precipitation; Lanes 5, 7: chlorophenol methanol precipitation; Lane 6: TCA precipitation (other supernatant precipitation data and cell pellet data are not shown)