Use of pIVEX plasmids for protein overproduction in Escherichia coli

Rogé, J; Betton, Jean‐Michel

doi:10.1186/1475-2859-4-18

Cited by 20 publications

(5 citation statements)

References 18 publications

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“…For CF expression of the GHSR, the encoding DNA was inserted into the pIVEX2.3d vector ( Rogé and Betton, 2005 ) with an AT-rich sequence at the 5’ end ( Haberstock et al, 2012 ). The maximum expression yield of about 1.4 mg per 1 ml reaction volume was obtained for the construct with the N-terminal SER-tag (sequence: KSSSSS, Figure 1 ), in comparison to 0.7 mg for the H-tag (sequence: KPYDGP), and 0.9 mg for the AT-tag (sequence: KYYKYY).…”

Section: Resultsmentioning

confidence: 99%

Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor

et al. 2020

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Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for 13 C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged 1 H- 13 C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly 13 C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions.

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Section: Resultsmentioning

confidence: 99%

Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor

et al. 2020

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“…The resulting recombinant plasmids were introduced into E. coli TOP 10 (Invitrogen) for sequence analysis and storage and into E. coli BL21λDE3/pDIA17 for protein expression. Induction was carried out with IPTG, as previously described [51] . Recombinant 6xHis-proteins were purified under denaturing conditions by affinity chromatography on Ni-NTA columns (Protino protein purification system, Macherey-Nagel, Düren, Germany) according to the manufacturers' recommendations.…”

Section: Methodsmentioning

confidence: 99%

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

et al. 2010

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Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection.

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“…Purified HET-s PFD can self-assemble in vitro into infectious and non-infectious polymorphic fibrillar forms 11 , 71 depending on aggregation conditions. The HET-s (218–289) gene fragment was incorporated into pIVEX vector 2.3 under the T7 promoter 72 . CF synthesis is a protein synthesis method performed in vitro without the requirement of living cells.…”

Section: Resultsmentioning

confidence: 99%

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis

et al. 2022

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Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.

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Use of pIVEX plasmids for protein overproduction in Escherichia coli

Cited by 20 publications

References 18 publications

Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor

Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis

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