An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies

Chhetri, Gaurav; Pandey, Tripti; Chinta, Ramesh; Kumar, Awanish; Tripathi, Timir

doi:10.1016/j.pep.2015.05.015

Cited by 19 publications

(12 citation statements)

References 51 publications

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“…3 A. Similarly, recombinant Aβ peptide fusion protein (∼30 kDa) showed increased fold of expression in the presence of 3% ethanol in comparison to a condition where ethanol was not present [10] ( Fig. 3 B).…”

Section: Methods Validationmentioning

confidence: 71%

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

2015

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Section: Methods Validationmentioning

confidence: 71%

“…Lane 3, Aβ induced in presence of 3% ethanol (+). Lane 4, Aβ induced in absence of 3% ethanol (−) [10] . (C) GlnRS protein: Lane 1, Molecular weight markers (kDa).…”

Section: Figmentioning

confidence: 99%

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

2015

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“…We obtained approximately $18 mg of puried Ab42 from 1 L of culture, which is a very high yield compared to the other strategies adapted for the affordable production of Ab peptide. 16,19,20,35 Characterization of the puried recombinant Ab42…”

Section: Expression and Purication Of The Mbp-ab42 Fusion Proteinmentioning

confidence: 99%

Highly efficient soluble expression, purification and characterization of recombinant Aβ42 fromEscherichia coli

et al. 2018

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Aggregation of amyloid-b protein (Ab) is hypothesized to be a seminal neuropathological event inAlzheimer's disease (AD). Recombinant expression and purification of Ab represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. Herein, we report a novel high-yield expression and purification method for Ab42 based on fusion with maltose binding protein Furthermore, the aggregates of the recombinant peptide had a strong cytotoxic effect on PC12 cells.The method described here can therefore be used to efficiently express the soluble fusion protein MBPAb42 and obtain high-purity Ab42 peptide, which can be used to understand the molecular mechanism of Ab42 fibrillization and screen new candidate drugs for AD.

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“…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 . Cheng et al also found that the addition of 4% ethanol has a 3-fold increase in production of 5AVA 10 .…”

mentioning

confidence: 99%

“…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 . 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 .…”

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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 improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies

Cited by 19 publications

References 51 publications

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

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

Highly efficient soluble expression, purification and characterization of recombinant Aβ42 fromEscherichia coli

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

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