Osmotic Stress Induced by Carbohydrates Enhances Expression of Foreign Proteins in Escherichia coli

Kagawa, Norio; Cao, Qing

doi:10.1006/abbi.2001.2516

Cited by 27 publications

(13 citation statements)

References 39 publications

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“…For example, 3A4 expression levels increased 31% in glycerol-fed batches (under pH control), compared to an 86% increase in the case of 2C9. The elevated P450 titers observed in glycerol-fed batches could not be attributed to enhanced growth, which was determined to be unaffected, but were most likely the result of improved expression and/ or protein folding, as proposed by Kagawa and Cao for 4A14 [14]. Without glycerol feeding and pH control, the pH of the fermentation medium in early stationary phase rose significantly, eventually reaching over pH 8.…”

Section: Fermentation Developmentmentioning

confidence: 84%

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Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Vail¹,

Homann²,

Hanna

et al. 2005

J IND MICROBIOL BIOTECHNOL

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Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase in Escherichia coli and used in the preparative synthesis of drug metabolites. Low dissolved oxygen (DO) concentration (<1%) during expression was found to be critical for producing active P450s. Control of temperature, pH and glycerol supplementation in 10-L fermentations enhanced enzyme expression 31-86%. Additional improvements were obtained by altering media formulations, resulting in bicistronic expression levels of 890, 1,800 and 1,010 nmol/L for 3A4, 2C9 and 1A2, respectively. The P450 titers achieved in fermentors exceeded those in flask fermentations by 3- to 6-fold in this study and up to 10-fold when compared with previously reported literature. Intact cells and isolated membranes obtained from 10-L fermentations were used to establish an efficient bioconversion system for the generation of metabolites. To demonstrate the utility of this approach, known metabolites of the anabolic steroid testosterone, the anti-inflammatory agent diclofenac and the analgesic agent phenacetin, were generated using 3A4, 2C9 and 1A2, respectively. The reaction conditions were optimized for pH, temperature, DO concentration, use of co-solvent and glucose supplementation. Conversion yields of 29-93% were obtained from 1-L reactions, enabling isolation of 59 mg 6beta-hydroxytestosterone, 110 mg 4'-hydroxydiclofenac and 88 mg acetaminophen.

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Section: Fermentation Developmentmentioning

confidence: 84%

“…Terrific broth, commonly reported for use with E. coli to express recombinant P450s [1,4,[12][13][14][15]23] had provided high P450 titers. Unfortunately, we discovered that use of different lots of this prepackaged complex medium resulted in up to 50% variation in enzyme synthesis while having little impact on culture growth.…”

Section: Fermentation Developmentmentioning

confidence: 99%

Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Vail¹,

Homann²,

Hanna

et al. 2005

J IND MICROBIOL BIOTECHNOL

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“…In addition, 2 g of arabinose was added to each 500 mL culture. 77 The cultures were grown for 44 h and then the cells were harvested by centrifugation. The cell pellet was resuspended in buffer 1 (100 mM potassium phosphate pH 7.4, 20% glycerol, 6 mM magnesium chloride, 0.1 mM dithiothreitol (DTT), and 0.2 mM phenylmethanesulfonylfluoride (PMSF), 1 mg each of DNase and RNase).…”

mentioning

confidence: 99%

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

et al. 2013

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CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N-terminus was used to express full length CYP2J2 in E. coli while maintaining wild-type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N-terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native-like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.

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“…Ethanol also enhances the expression of a P450 and a recombinant b-galactosidase in E. coli (Thomas & Baneyx 1996) and chloramphenicol and tetracycline also enhance the expression of several P450s (Kusano et al 1999). Arabinose and other sugars also enhanced the expression of P450 genes in E. coli (Kagawa & Cao 2001).…”

Section: Introductionmentioning

confidence: 97%

Expression of an antibody-targeted plasminogen activator in Escherichia coli using chemically induced stress responses

2004

Biotechnol Lett

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A recombinant gene coding for an antibody-targeted urokinase-type plasminogen activator was constructed for the purpose of enhancing the thrombolytic specificity of urokinase. The recombinant gene was cloned into prokaryotic expression vector pTrcHisA, and transformed into Escherichia coli strain Rosetta (DE3). Less than 4 mg of the desired protein l(-1) could be obtained in the form of inclusion bodies. Of various inducers and enhancers of stress responses, the heat-shock enhances, streptomycin, the osmotic stress inducers, D-arabinose and sucrose, and the cold-shock enhancer, tetracycline, simulated the expression of the antibody-targeted plasminogen activator by 2-5-fold.

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Osmotic Stress Induced by Carbohydrates Enhances Expression of Foreign Proteins in Escherichia coli

Cited by 27 publications

References 39 publications

Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

Expression of an antibody-targeted plasminogen activator in Escherichia coli using chemically induced stress responses

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