GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli

Sahu, Santosh Kumar; Rajasekharan, Archita; Gummadi, Sathyanarayana N.

doi:10.1007/s10529-009-0073-7

Cited by 14 publications

(9 citation statements)

References 22 publications

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“…The addition of ethanol to the culture medium was tested for molecular chaperone induction and recombinant protein stabilization Baneyx 1996, 1997). In contrast with a previous study that showed an optimal ethanol concentration of 3% (Sahu et al 2009), yields from the present system were increased 2-fold after addition of 1% ethanol ( Figure 4D). To the best of our knowledge, this is the first report to demonstrate the effects of ethanol supplementation on CYP expression in E. coli.…”

Section: Discussioncontrasting

confidence: 99%

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Uchida

Akashi

Aoki

2015

Plant Biotechnology

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Biochemical analyses of metabolic enzymes are performed using in vitro assays with enzymes and substrates. Enzyme samples are generally prepared from heterologous cell expression systems, which are also used as tools to obtain substrates that are not commercially available or cannot be easily isolated from natural sources. Cytochrome P450 (CYP) comprises a widely distributed family of monooxygenases that are commonly used for biosynthesis of natural products. Since CYP activity requires an electron transport system with membrane bound CYP reductase (CPR), it has been believed that CYP is not easily expressed in Escherichia coli, despite multiple advantages as a heterologous system compared with other systems that employ eukaryotic yeast and insect cells. In this study, we demonstrated simple and efficient methods for functional expression of CYPs in E. coli using commercially available vectors in which the transmembrane-domain truncated CYP was co-expressed with CPR as a discrete polypeptide, and we also used them to identify CYP81E11, CYP81E12, and CYP81E18 of soybean as isoflavone 2′-hydroxylase. Culture conditions were optimized for the bioconversion of I2′H, and the highest production was 161 mg l −1 of medium under optimized conditions. Subsequently, six other CYPs that are involved in flavonoid biosynthesis were tested for their applicability in the E. coli expression system. Establishment of the present method may facilitate functional expression of CYPs for preparation of CYP products, including substrates for enzymatic reactions, valuable natural products, and their unnatural derivatives.

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Section: Discussioncontrasting

confidence: 99%

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Uchida

Akashi

Aoki

2015

Plant Biotechnology

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“…Trigger factor, on the other hand, associates with the synthesized proteins as soon as they leave ribosome and through its interaction with their exposed hydrophobic patches averts their subsequent aggregation [14], [38]. Co-expression of molecular chaperones resulted in enhanced solubility and production of recombinant rice plant catalase A [39] active ribonuclease inhibitor [40], human scramblase 1 [41] and zeta-crystallin [42]. Solubility of his-tagged ALDH3A1 was significantly improved under conditions of co-expressing the pG-KJE8) suggesting that dnaK/dnaJ/grpE and groES/groEL are the essential chaperones for the correct folding of recombinant human ALDH3A1 (his-tagged ALDH3A1) when over-expressed in E. coli .…”

Section: Discussionmentioning

confidence: 99%

Efficient E. coli Expression Strategies for Production of Soluble Human Crystallin ALDH3A1

et al. 2013

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Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we compared different E. coli expression strategies (namely the maltose binding protein; MBP- and the 6-his-tagged expression systems) under conditions of auto-induction and co-expression with E. coli’s molecular chaperones where appropriate. Thus, we aimed to screen the efficiency of these expression strategies in order to improve solubility of recombinant ALDH3A1 when expressed in E. coli. We showed that the MBP- tagged expression in combination with lower-temperature culture conditions resulted in active soluble recombinant ALDH3A1. Expression of the fused 6-his tagged-ALDH3A1 protein resulted in poor solubility and neither lowering temperature culture conditions nor the auto-induction strategy improved its solubility. Furthermore, higher yield of soluble, active native form of 6-his tagged-ALDH3A1 was facilitated through co-expression of the two groups of E. coli’s molecular chaperones, GroES/GroEL and DnaK/DnaJ/GrpE. Convenient one step immobilized affinity chromatography methods were utilized to purify the fused ALDH3A1 hybrids. Both fusion proteins retained their biological activity and could be used directly without removing the fusion tags. Taken together, our results provide a rational option for producing sufficient amounts of soluble and active recombinant ALDH3A1 using the E. coli expression system for conducting functional studies towards elucidating the biological role(s) of this interesting corneal crystallin.

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“…There is another degree of complication that the successful expression of the target gene in its desirable or soluble form still does not guarantee its activity. To overcome such difficulties and produce the target proteins successfully, various methods have been developed: strains capable of translating rare codons, strains capable of forming disulfide bonds intracellularly, plasmids with a specifically designed promoter, tags or fusion partners conferring higher expression and/or solubility and/or simpler purification, expression with molecular chaperones, expression at lower temperatures, and secretion to periplasm or medium . With all these available measures, one has to go through one by one until the problem is solved, but there is also a greater possibility that none of those will work.…”

Section: Introductionmentioning

confidence: 99%

Relationship between Protein Expression Pattern and Host Metabolome Perturbation as Monitored by Two‐Dimensional NMR Spectroscopy

Chae

Kim

2019

Bulletin Korean Chem Soc

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In producing large amounts of heterologous proteins, researchers most often use Escherichia coli as a host thanks to its extensively studied genetics, simple growth procedure, and low cost. However, the desired protein is often produced only in a form of inclusion bodies. Researchers have tried to devise a way to circumvent such a problem, and the ones using fusion partners seem to be the most successful. Based on our previous observation that the host metabolome was related to the outcomes of protein expression patterns, we proceeded to perturb the metabolome by applying a salt stress to see if we could shake up metabolite compositions to make them better suited for soluble expression of the target protein. We examined a subset of the metabolites which had been partially labeled with input 13C‐glucose. We tested 11 genes under 4 different NaCl concentrations, and identified 18 metabolites using the heteronuclear single quantum coherence NMR experiment. Most of the proteins kept their expression profiles unchanged, but two proteins were converted from inclusion bodies to a soluble form with increased NaCl concentration. Through the statistical analysis, we could identify a region where the soluble protein production was favored in the metabolite space. We hope that this work would provide an alternative strategy to produce the recombinant proteins in their soluble or native forms, not only in E. coli but also in other hosts.

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GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli

Cited by 14 publications

References 22 publications

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Efficient E. coli Expression Strategies for Production of Soluble Human Crystallin ALDH3A1

Relationship between Protein Expression Pattern and Host Metabolome Perturbation as Monitored by Two‐Dimensional NMR Spectroscopy

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