Membrane protein expression and production: effects of polyhistidine tag length and position

Mohanty, Arun; Wiener, Michael C.

doi:10.1016/j.pep.2003.10.010

Cited by 113 publications

(60 citation statements)

References 45 publications

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“…Moreover, it causes low interference during the crystallization process, suggesting that His-tagged fusion proteins can be used for direct structural analysis (Bucher et al, 2002;Mohanty & Wiener, 2004;Reyes et al, 2006). Thus, the His-tagged P9-1 protein obtained in this study was suitable for the following structural analysis.…”

Section: Expression and Purification Of P9-1mentioning

confidence: 88%

Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

Zhang

Liu

Liu³

et al. 2008

Journal of General Virology

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Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a Histagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, a-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.

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Section: Expression and Purification Of P9-1mentioning

confidence: 88%

Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

Zhang

Liu

Liu³

et al. 2008

Journal of General Virology

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“…It included a longer His-tag (eight instead of six residues), which had been reported to improve binding to the metal-affinity resin in other cases [23]. This construct (E 140 A HtpX-2kDa-tag) was next cloned into modified pETM30 and pET28 expression vectors comprising a collection of N-terminal fusion proteins removable by TEV peptidase.…”

Section: Screening For the Best Expression And Purification System Fomentioning

confidence: 99%

Expression and purification of integral membrane metallopeptidase HtpX

Arolas

García‐Castellanos

Goulas

et al. 2014

Protein Expression and Purification

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The authors state they have no competing financial interest Arolas et al. ! 2! Highlights• The integral membrane metallopeptidase HtpX was overexpressed in E. coli.• A catalytically ablated HtpX mutant was designed to avoid self-cleavage.• The protein was extracted from membranes and purified in octyl-!-D-glucoside.• The protein was purified by cobalt-affinity, anion-exchange and size-exclusion.• The overall system renders milligram amounts of HtpX and fosters structural studies. AbstractLittle is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli.Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His 8 -tag, extracted from the membranes using octyl-!-D-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and sizeexclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members.

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“…Similar purification tactics have been found to work well for the isolation of membrane proteins (David et al, 1997;Feng et al, 2002;Grisshammer and Tucker, 1997;Mohanty and Wiener, 2004;Theis et al, 2001). However, flow cytometry does not lend itself well to the rapid sorting and isolation of high-expressing cells that do not have a quantifiable tag on the expressed protein.…”

Section: Selection Of High-expressing Yeast Cellsmentioning

confidence: 99%

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

O’Malley

Lazarova

Britton

et al. 2007

Journal of Structural Biology

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The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A 2 a receptor fused to a green fluorescent protein (A 2 aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A 2 aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A 2 aR and A 2 aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A 2 aR-GFP-His 10 . One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A 2 aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A 2 a-His 10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A 2 aR yet achieved from any heterologous expression system.

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Membrane protein expression and production: effects of polyhistidine tag length and position

Cited by 113 publications

References 45 publications

Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

Expression and purification of integral membrane metallopeptidase HtpX

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

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