Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology
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This paper reported the optimization of expression of the cloned human B lymphocyte stimulator (hBLyS) gene in Escherichia coli strain BL21 (DE3) by using lactose as the inducer. It was found that expression level of hBLyS achieved the highest after the BL21 (DE3) had been induced for 5 h with 0.1 g/L lactose begining from strain density of OD_(600) = 1.0. The expression level of hBLyS protein was about 15.7% of the total cellular proteins. The inducing efficiency of lactose was lower than IPTG (isopropyl-β-D-thiogalactopyranoside), but because of the toxicity and the high cost of the latter, lactose is a promising inducer in the lac-promotor based expression system.
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Objective To study the influence of various inducers on the expression of recombinant HSP-MUC1 protein in E.coli BL21(DE3) by fermentation and optimize the condition for expression.Methods Express recombinant HSP-MUC1 protein by fermentation using IPTG and lactose as inducers respectively,and analyze the expression level and character of HSP-MUC1.Results The bacteria using IPTG as an inducer could be broken by common homogenization.However,the bacteria using lactose as inducer could not be broken by the same method.As the increasing time for expression,the expression level of HSP-MUC1 protein using lactose was comparable to that using IPTG as inducer.Conclusion Lactose might be an ideal inducer for the expression of recombinant protein.
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Lactose permease
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Lactose is not itself an inducer of the lac operon, nor is it converted to an inducer by ebg+ beta-galactosidase of Escherichia coli. We report here the isolation of a mutant Ebg beta-galactosidase which is capable of converting lactose into an inducer of the lac operon.
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L-arabinose operon
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According to the amino acid sequence of Monellin and the bias in coden choice in Escheria coli,a single chain monellin gene was synthesized and subcloned into a Escheria coli expression vector pET-28a. The recombined plasmid pET28a-mon was then transformed into Escheria coli BL21(DE3).Lactose was used as an inducer instead of IPTG. Through proper optimization of induction conditions,an expression level 33.09% of total cellular protein was achieved.The expression level of recombinant protein induced by lactose was basically the same as that induced by IPTG. The result indicated that lactose could be used as a promising inducer in the production of recombined sweet protein Monellin.
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N-acetylornithine deacetylase was expressed in recombinant bacteria BL21(DE3)-pET22b-argE.The location of the target protein was determined.The effects of induction temperature,type of inducer concentration of inducer,initial cell density,induction time on the activity expression of the target protein were studied.The results showed that both IPTG and lactose could be inducers,and the induction effect of lactose was better than that of IPTG.With the optimized active inducing condition of adding 15 g/L lactose at initiated OD600 of 0.46 and cultivating for 18 h at 20 ℃,the NAOase activity was increased by 168 folds from initial 1.68U/mL to 182.99U/mL.
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Abstract The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose‐inducible promoters P tac and P trc by the natural inducer lactose and the synthetic inducer isopropyl‐β‐ d ‐thiogalactopyranoside (IPTG) was investigated. Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall. In E. coli strains lacking a functional LacY, IPTG is required for induction of P tac and P trc . In E. coli strains carrying a functional LacY, induction of P trc and P tac with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used. In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY + strains.
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