The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli

Singh, Jas; Whitwill, Steve; Lacroix, Geneviève; Douglas, Jennifer L.; Dubuc, Elyse; Allard, Ghislaine; Keller, W. A.; Schernthaner, Johann P.

doi:10.1016/j.pep.2009.04.003

Cited by 17 publications

(21 citation statements)

References 53 publications

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“…Toward this aim we first analyzed the primary sequences characteristics of a family of plant proteins known as dehydrins, because they have been shown to be intrinsically disordered, to have potential chaperone activity (42–45), and to function as both an anti-aggregant and an enzyme preservation agent (31, 32, 46–54). Moreover, two family members have been shown to solubilize membrane proteins identified as recalcitrant to overexpression (55). Table 1 shows the compilation of characteristics of both the disorder and solubility predictions for the six known A. thaliana dehydrin proteins.…”

Section: Resultsmentioning

confidence: 99%

Sweeping Away Protein Aggregation with Entropic Bristles: Intrinsically Disordered Protein Fusions Enhance Soluble Expression

et al. 2012

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Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can enable the target protein to fold free from interference. Using both naturally-occurring and artificial polypeptides we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have low sequence complexity and high net charge, but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently-used fusions such as maltose-binding-protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physio-chemical properties they did not perturb the structure, conformational stability nor function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically-disordered fusions, and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

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Section: Resultsmentioning

confidence: 99%

Sweeping Away Protein Aggregation with Entropic Bristles: Intrinsically Disordered Protein Fusions Enhance Soluble Expression

et al. 2012

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“…Apart for agronomical purposes, LEA proteins might be useful for other biotechnological applications in relation to their capacity to prevent formation of aggregated proteins. For instance, fusion of a truncated peptide of BNECP63 (PF02987) to target proteins is sufficient to provide a recombinant intrinsic membrane protein and hepatitis C viral protein in a soluble form or outside of inclusion bodies in E. coli [93]. This experiment demonstrates the use of LEA-type peptides in facilitating the production of recombinant proteins.…”

Section: Lea Proteinsmentioning

confidence: 88%

Desiccation tolerance: From genomics to the field

2010

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“…Although a variety of LEA proteins exist, all show has hydrophilic character [12]. Several studies have reported on improvements in the solubility of protein expressed in cell body achieved by utilizing the hydrophilic characteristic of LEA proteins [13],[14],[15]. Singh et al developed a technique for facilitating recombinant expression of recalcitrant proteins by using LEA protein as fusion partners [13].…”

Section: Introductionmentioning

confidence: 99%

“…Several studies have reported on improvements in the solubility of protein expressed in cell body achieved by utilizing the hydrophilic characteristic of LEA proteins [13],[14],[15]. Singh et al developed a technique for facilitating recombinant expression of recalcitrant proteins by using LEA protein as fusion partners [13]. They found that fusion of the LEA protein provides sufficient solubility to permit overexpression of hydrophobic protein in E.coli .…”

Section: Introductionmentioning

confidence: 99%

Boost Protein Expression through Co-Expression of LEA-Like Peptide in Escherichia coli

Ikeno

Haruyama

2013

PLoS ONE

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The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

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The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli

Cited by 17 publications

References 53 publications

Sweeping Away Protein Aggregation with Entropic Bristles: Intrinsically Disordered Protein Fusions Enhance Soluble Expression

Sweeping Away Protein Aggregation with Entropic Bristles: Intrinsically Disordered Protein Fusions Enhance Soluble Expression

Desiccation tolerance: From genomics to the field

Boost Protein Expression through Co-Expression of LEA-Like Peptide in Escherichia coli

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