An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli

Chhetri, Gaurav; Kalita, Parismita; Tripathi, Timir

doi:10.1016/j.mex.2015.09.005

Cited by 59 publications

(34 citation statements)

References 25 publications

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“…Removing the native signal peptide from the PhaZ sequence is a key step in diminishing the formation of inclusion bodies and avoiding completely insoluble PhaZs when using pET‐22b(+) or plasmids that add signal sequences. Induction at higher IPTG concentrations and addition of ethanol in the induction stage (Chhetri, Kalita, & Tripathi, ) did not lead to improved expression.…”

Section: Resultsmentioning

confidence: 99%

“…is a key step in diminishing the formation of inclusion bodies and avoiding completely insoluble PhaZs when using pET-22b(+) or plasmids that add signal sequences. Induction at higher IPTG concentrations and addition of ethanol in the induction stage(Chhetri, Kalita, & Tripathi, 2015) did not lead to improved expression.The presence of the C-terminal His-tag was verified through Western blot for SF and IF of PhaZ Cte and PhaZ Mal before proceeding to large-scale inductions for purification. An example is shown in Figure 1b, c for the SF and IF of PhaZ Cte induced at 15°C with 1 mM IPTG.…”

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confidence: 82%

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Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

et al. 2020

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Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His‐tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate‐based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

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Section: Resultsmentioning

confidence: 99%

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confidence: 82%

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

et al. 2020

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“…One way to optimize the level of expression is by manipulating the composition of the culture medium. In addition to the addition of isopropyl β-D-1thiogalactopyranoside (IPTG) to the media, ethanol is also known to increase the recombinant protein expression (Chhetri, et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Effect of Ethanol and IPTG on the Recombinant Jembrana Trans-Activator of Transcriptation Protein Expression

et al. 2018

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Jembrana diseases are caused by Jembrana Diseases Virus (JDV). The previous study showed that Jembrana Trans-Activator of Trancriptation (JTAT) recombinant protein is effective as a vaccine for Jembrana diseases. The production of JTAT protein needs to be optimized to obtain a higher amount of vaccine. High expression of JTAT protein will produce a high vaccine product. This study aimed to examine the effect of the addition of ethanol and IPTG in E. coli media on the expression of JTAT recombinant protein. This research was experimental research with factorial RAL design with a variation factor of ethanol and IPTG. Qualitatively, the induction of each IPTG, ethanol and interaction between the two could induce the expression of JTAT protein and could be identified with SDS-PAGE at ±11.8 kDa. Statistically, the induction of IPTG, ethanol and interaction between the two were not significantly different. Qualitative and quantitative data show that ethanol can induce JTAT protein expression. This result can be used as a preliminary study to test the effectiveness of ethanol as a substitute for IPTG in inducing the recombinant protein expression.

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“…The expression of proteins is often induced by isopropyl β-D-thiogalactopyranoside (IPTG) in strain expression system. However, the expression level of proteins is still relatively low 24 . Effective strategies for improving expression level of proteins were developed, include the optimization of the host strains, vector, culture medium and gene sequences, as well as the addition of ethanol 25 .…”

mentioning

confidence: 99%

“…Effective strategies for improving expression level of proteins were developed, include the optimization of the host strains, vector, culture medium and gene sequences, as well as the addition of ethanol 25 . Ethanol has been demonstrated to works with T7 or T5 promoters, and a relatively higher level of protein expression was observed with the addition of 2%-4% ethanol 24 . In Chhetri' research, the addition of 3% ethanol could not only improve the expression level of the heterogeneous amyloid-beta peptide 25 , but also increase the enzymatic activity by 5-fold 26 .…”

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confidence: 99%

Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment

Cheng

Luo

Duan

et al. 2020

Sci Rep

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Microorganisms can utilize biomass to produce valuable chemicals, showing sustainable, renewable and economic advantages compared with traditional chemical synthesis. As a potential five-carbon platform polymer monomer, 5-aminovalerate has been widely used in industrial fields such as clothes and disposable goods. Here we establish an efficient whole-cell catalysis for 5-aminovalerate production with ethanol pretreatment. In this study, the metabolic pathway from L-lysine to 5-aminovalerate was constructed at the cellular level by introducing L-lysine α-oxidase. The newly produced H 2 o 2 and added ethanol both are toxic to the cells, obviously inhibiting their growth. Here, a promising strategy of whole-cell catalysis with ethanol pretreatment is proposed, which greatly improves the yield of 5-aminovalerate. Subsequently, the effects of ethanol pretreatment, substrate concentration, reaction temperature, pH value, metal ion additions and hydrogen peroxide addition on the wholecell biocatalytic efficiency were investigated. Using 100 g/L of L-lysine hydrochloride as raw material, 50.62 g/L of 5-aminovalerate could be excellently produced via fed-batch bioconversion with the yield of 0.84 mol/mol. The results show that a fast, environmentally friendly and efficient production of 5-aminovalerate was established after introducing the engineered whole-cell biocatalysts. This strategy, combined with ethanol pretreatment, can not only greatly enhance the yield of 5-aminovalerate but also be applied to the biosynthesis of other valuable chemicals.

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An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli

Abstract: Graphical abstract

Cited by 59 publications

References 25 publications

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

Effect of Ethanol and IPTG on the Recombinant Jembrana Trans-Activator of Transcriptation Protein Expression

Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment

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