Impact of oxygen supply on rtPA expression in Escherichia coli BL21 (DE3): ammonia effects

Wang, Hengwei; Wang, Fengqing; Wei, Dongzhi

doi:10.1007/s00253-008-1756-z

Cited by 12 publications

(12 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The effects of varying the concentrations of yeast extract and bacto tryptone in TB were also investigated, but again no increases in SELP production levels were noted. Ammonium and amino acid additives were then examined as previous studies have indicated the positive effects of ammonium as a nitrogen source, even in the presence of excess organic nitrogen [32]. Furthermore, due to the highly repetitive nature of the amino acid sequences of PBPs, the potential benefits of exogenously added amino acids can be easily understood and has been previously shown.…”

Section: Resultsmentioning

confidence: 99%

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

et al. 2013

View full text Add to dashboard Cite

BackgroundSilk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.ResultsThis study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.ConclusionsWe have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.

show abstract

Section: Resultsmentioning

confidence: 99%

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

et al. 2013

View full text Add to dashboard Cite

show abstract

“…It is well documented that recombinant protein expression in E. coli cells is decreased by acetate accumulation [5] , [6] . However, our previous study showed that acetate concentrations up to 200 mM had little effect on the protein expression of recombinant tissue plasminogen activator (rtPA) in this strain [2] .…”

Section: Introductionmentioning

confidence: 85%

“…The use of glucose or glycerol as the carbon source in a complex medium may result in acetate accumulation at high levels, often up to 50–100 mM, particularly in high-density fermentations [1] , [2] . In aqueous solution, the acetate anion (Ac – ) exists in equilibrium with the undissociated acetic acid (HAc) and their relative quantities depend on pH.…”

Section: Introductionmentioning

confidence: 99%

“…However, when a complex medium is used to express recombinant proteins, carbon source starvation seldom occurs and acetate transport mainly depends on the free diffusion of HAc across the cell membrane [3] . In scale-up of bioprocesses from shake flasks to fermentors, acetate production tends to increase and is usually regarded as a cause of the decline in protein expression [2] , [5] , [6] .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Wang

et al. 2014

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

show abstract

“…The plasmid pKD46 (Amp R ) was used as a helper plasmid for increasing the efficiency of homologous recombination, and pCP20 (Cm R /Amp R ) was used for eliminating the resistance marker of the mutants in the construction of leaky strains (Datsenko and Wanner, ). The plasmids pET32a‐ pth and pET22b‐ rpa , both constructed without signal sequences, were used for the expression of exogenous proteins Trx‐hPTH (a thioredoxin fusion protein used for its residence at the periplasmic space of E. coli ) (Bayer et al ., ; Fu et al ., ) and rPA (Wang et al ., ) in periplasmic space or in cytoplasm. The primers were designed by Primer Premier 5.0 and verified by Oligo 7.0.…”

Section: Methodsmentioning

confidence: 99%

Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli

Chen

Cao

Xie

et al. 2014

Microbial Biotechnology

View full text Add to dashboard Cite

In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l−1.

show abstract

Impact of oxygen supply on rtPA expression in Escherichia coli BL21 (DE3): ammonia effects

Cited by 12 publications

References 42 publications

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli

Contact Info

Product

Resources

About