Abstract:A novel air separation system based on permeable membrane gas separation technology was used to cultivate Escherichia coli. The system fulfilled the dissolved oxygen requirements of a culture of E. coli grown on a glucose synthetic medium at a high and constant growth rate of 0.55 h-'. A biomass yield of 45 g (dry weight) per liter was achieved, and no by-product inhibition by acetate or C02 was observed.
“…In the first step, a single colony of transformed E. coli was transferred from TYE (tryptone yeast extract) medium [bactotryptone (10 g/l), yeast extract (5 g/l), NaCl (8 g/l) and bacteriological agar (15 g/l), pH 7.5] into 10 ml of 2×TYE medium [bactotryptone (16 g/l), yeast extract (10 g/l) and NaCl (5 g/l), pH 7.0] and incubated in a 250 ml shake flask at 37 °C for 10 h at 150 rev./min. In the second step, 0.5 ml of first step inoculum was used to inoculate 50 ml of a medium [16] containing glucose (10 g/l), yeast extract (1 g/l), K 2 HPO 4 (10 g/l), KH 2 PO 4 (13 g/l), (NH 4 ) 2 HPO 4 (3 g/l), NaH 2 PO 4 (4.6 g/l), MgSO 4 ·7H 2 O (0.46 g/l) and 10 ml/l trace‐salt solution [CuSO 4 ·5H 2 O (2 mg/l), Al 2 (SO 4 ) 3 (10 mg/l), MgCl 2 .4H 2 O (20 mg/l), Na 2 MoO 4 ·2H 2 O (50 mg/l), H 3 BO 3 (1 mg/l), CoCl 2 (2.5 g/l), ZnSO 4 (5 mg/l), FeSO 4 (50 mg/l) and NiCl 2 ·6H 2 O (1 mg/l)] along with antibiotics [ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml)]. This second‐step inoculum in a 500 ml shake flask was incubated at 37 °C for 6 h at 220 rev./min and used for seeding the bioreactor.…”
A purification method employing a process-control strategy was developed for improving the yield of rhG-CSF (recombinant human granulocyte colony-stimulating factor). A purity of >/=99% with an overall yield of 2.18 g/l was achieved in the present study. Analysis of the product during purification indicated that detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs (host cell proteins) without removing nucleic acid. Cysteine concentration was a key parameter in protein refolding. The bed height and HETP (height equivalent theoretical plates) value in the SEC (size-exclusion chromatography) column was evaluated and its impact on the resolution was studied. Formulation during SEC was found to be crucial for increasing the product yields with saving of time and process costs. The yield obtained in the present study is nearly four times higher than that reported in the literature. The product obtained was found to be acceptable for toxicological studies.
“…In the first step, a single colony of transformed E. coli was transferred from TYE (tryptone yeast extract) medium [bactotryptone (10 g/l), yeast extract (5 g/l), NaCl (8 g/l) and bacteriological agar (15 g/l), pH 7.5] into 10 ml of 2×TYE medium [bactotryptone (16 g/l), yeast extract (10 g/l) and NaCl (5 g/l), pH 7.0] and incubated in a 250 ml shake flask at 37 °C for 10 h at 150 rev./min. In the second step, 0.5 ml of first step inoculum was used to inoculate 50 ml of a medium [16] containing glucose (10 g/l), yeast extract (1 g/l), K 2 HPO 4 (10 g/l), KH 2 PO 4 (13 g/l), (NH 4 ) 2 HPO 4 (3 g/l), NaH 2 PO 4 (4.6 g/l), MgSO 4 ·7H 2 O (0.46 g/l) and 10 ml/l trace‐salt solution [CuSO 4 ·5H 2 O (2 mg/l), Al 2 (SO 4 ) 3 (10 mg/l), MgCl 2 .4H 2 O (20 mg/l), Na 2 MoO 4 ·2H 2 O (50 mg/l), H 3 BO 3 (1 mg/l), CoCl 2 (2.5 g/l), ZnSO 4 (5 mg/l), FeSO 4 (50 mg/l) and NiCl 2 ·6H 2 O (1 mg/l)] along with antibiotics [ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml)]. This second‐step inoculum in a 500 ml shake flask was incubated at 37 °C for 6 h at 220 rev./min and used for seeding the bioreactor.…”
A purification method employing a process-control strategy was developed for improving the yield of rhG-CSF (recombinant human granulocyte colony-stimulating factor). A purity of >/=99% with an overall yield of 2.18 g/l was achieved in the present study. Analysis of the product during purification indicated that detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs (host cell proteins) without removing nucleic acid. Cysteine concentration was a key parameter in protein refolding. The bed height and HETP (height equivalent theoretical plates) value in the SEC (size-exclusion chromatography) column was evaluated and its impact on the resolution was studied. Formulation during SEC was found to be crucial for increasing the product yields with saving of time and process costs. The yield obtained in the present study is nearly four times higher than that reported in the literature. The product obtained was found to be acceptable for toxicological studies.
“…The results of prior experiments in our laboratory (both in vitro experiments and animal experiments) were used to decide sample size for each of the experiments shown. 37 – 41 Fig. 2 Cytokine-induced TNF release inhibited by MECO-1.…”
E. coli releases a 33 amino acid peptide melanocortin-like peptide of E. coli (MECO-1) that is identical to the C-terminus of the E. coli elongation factor-G (EF-G) and has interesting similarities to two prominent mammalian melanocortin hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH). Note that MECO-1 lacks HFRW, the common pharmacophore of the known mammalian melanocortin peptides. MECO-1 and the two hormones were equally effective in severely blunting release of cytokines (HMGB1 and TNF) from macrophage-like cells in response to (i) endotoxin (lipopolysaccharide) or (ii) pro-inflammatory cytokine HMGB-1. The in vitro anti-inflammatoty effects of MECO-1 and of alpha-MSH were abrogated by (i) antibody against melanocortin-1 receptor (MC1R) and by (ii) agouti, an endogenous inverse agonist of MC1R. In vivo MECO-1 was even more potent than alpha-MSH in rescuing mice from death due to (i) lethal doses of LPS endotoxin or (ii) cecal ligation and puncture, models of sterile and infectious sepsis, respectively.
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