High-level Expression of Cecropin X in Escherichia coli

Abstract

Cecropin X is a short cationic peptide with a broad antibacterial and antitumor spectrum. Here, we report the production of a tumor necrosis factor (TNFα)-cecropin X fusion protein under the control of a temperature-inducible P R promoter in the bacterial expression vector pRC. During fermentation, we studied and optimized essential parameters including the type of host cells, medium, timing of induction, post-induction time and dissolved oxygen level. Using the suitable conditions in the fermentation, up to 20 % ~ 23 % of the total cellular proteins is produced as the fusion protein, mostly in the form of inclusion bodies. After washing, on average about 5.27 g dried inclusion bodies could be collected from 1 L broth and the purity of inclusion bodies reached 80 %. Cecropin X obtained by cleaving the fusion protein with cyanogen bromide showed remarkable tumorcidal activity against mouse Lewis lung carcinoma 3LL in vivo.

Statistical optimization of culture medium for the overproduction of chicken anemia virus immunogen‐ VP1 protein in a recombinant E. coli for vaccine application

“…The fact that the glucose had little effect on VP1 protein production might be a result of catabolite repression. When a high concentration of glucose is used during E. coli cultivation, this abolishes catabolite repressor protein activity, which is how this protein senses the level of cyclic AMP in cells . Shen et al indicated that the presence of an additional carbon source, such as glycerol or glucose, might be beneficial to cell growth.…”

Insect natural products and processes: New treatments for human disease

“…This cost can be reduced by using truncated derivatives and recombinant technology , although production in bacteria has been hindered by their anti-bacterial activity and proteolytic degradation (Bommarius et al, 2010). Good progress, however, has been reported using recombinant technology to scale up the production of cecropin for clinical trials (Shen et al, 2007;Chen et al, 2009b). Chen et al (2009b) obtained highly efficient expression of recombinant cecropin AD in B. subtilis yielding a pure, highly stable, peptide with potent antimicrobial activity at low MICs (minimal inhibitory concentrations) and applicable to industrial production.…”

Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula

“…After optimizing the time and concentration of IPTG induction, the different medium cultures were investigated with (1) LB medium: 0.5% yeast extract, 1% peptone and 1% NaCl and 1.5% agar (Vulfson et al, 2001), (2) SOB medium culture: 2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 and 10 mM MgSO4 (Shen et al, 2007), (3) SOC medium culture: 2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose (Shen et al, 2007), (4) HSG medium culture: 1.49% glycerol, 0.7% yeast extract, 1.35% tryptone, 0.014% MgSO 4 .H 2 O, 0.15% KH 2 PO 4 , 0.23% K 2 HPO 4 and 0.5% (Miksch et al, 2008), and (6) TB medium culture: 1.2% peptone, 2.4% yeast extract, 72mM K 2 HPO 4 , 17mM KH 2 PO 4 and 0.4% glycerol (Shen et al, 2007) supplemented with 1% glucose and 100 μg/ml ampicillin. The culture was carried out on a rotation shaker with a speed of 200 rpm to an OD600 of 0.8.…”