One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent

Wang, Lili; Zhou, Hu; Li, Zhengjun; Lim, Teck Kwang; Lim, Xin Shan; Lin, Qingsong

doi:10.1016/j.pep.2015.08.014

Cited by 7 publications

(13 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The expression vector pET H6-AqpZ encodes fulllength E. coli aqpz gene (GenBank ID U38664.1) with an N-terminal 6×His tag sequence of MGSSHHHHHHEF. 29 The wt-and Δ4-AqpZ plasmids were expressed in E. coli BL21 (DE3) strain by a modified "dual media" approach. 30 Briefly, several expression colonies were inoculated into 100 mL of LB medium with 100 μg/mL ampicillin for cultivation expansion overnight.…”

Section: ■ Experiments and Methodsmentioning

confidence: 99%

“…Water permeability of wtand Δ4-AqpZ POPC/POPG proteoliposomes was characterized as described in the literature. 29 In detail, the POPC/POPG blank liposomes and AqpZ and Δ4-AqpZ proteoliposomes (lipid/protein ratio of 10, w/w) were prepared by dialysis following the same procedure used to prepare the samples for NMR experiments. The water permeability of liposomes and proteoliposomes was measured by an SX20 stopped-flow spectrometer (Applied Photophysics, Leatherhead, UK) at a light source of 577 nm wavelength.…”

Section: ■ Experiments and Methodsmentioning

confidence: 99%

“…The Δ4-AqpZ (R3K, R75K, R224K, R230K) mutant was constructed by site-directed-mutagenesis of wild-type (wt) pET H6-AqpZ plasmid. The expression vector pET H6-AqpZ encodes full-length E. coli aqp z gene (GenBank ID U38664.1) with an N-terminal 6×His tag sequence of MGSSHHHHHHEF . The wt- and Δ4-AqpZ plasmids were expressed in E. coli BL21 (DE3) strain by a modified “dual media” approach .…”

Section: Experiments and Methodsmentioning

confidence: 99%

“…Water permeability of wt- and Δ4-AqpZ POPC/POPG proteoliposomes was characterized as described in the literature . In detail, the POPC/POPG blank liposomes and AqpZ and Δ4-AqpZ proteoliposomes (lipid/protein ratio of 10, w/w) were prepared by dialysis following the same procedure used to prepare the samples for NMR experiments.…”

Section: Experiments and Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Gating Mechanism of Aquaporin Z in Synthetic Bilayers and Native Membranes Revealed by Solid-State NMR Spectroscopy

et al. 2018

Self Cite

View full text Add to dashboard Cite

Aquaporin Z (AqpZ) is an integral membrane protein that facilitates transport of water across Escherichia coli cells with a high rate. Previously, R189, a highly conserved residue of the selective filter of AqpZ, was proposed as a gate within the water channel on the basis of the observation of both open and closed conformations of its side chain in different monomers of an X-ray structure, and the observation of rapid switches between the two conformations in molecular dynamic simulations. However, the gating mechanism of the R189 side chain remains controversial since it is unclear whether the different conformations observed in the X-ray structure is due to different functional states or is a result of perturbation of non-native detergent environments. Herein, in native-like synthetic bilayers and native E. coli membranes, a number of solid-state NMR techniques are employed to examine gating mechanism of the R189 side chain of AqpZ. One R189 side-chain conformation is highly evident since only a set of peaks corresponding to the R189 side chain is observed in 2D N-C spectra. The immobility of the R189 side chain is detected by H-N dipolar lineshapes, excluding the possibility of the rapid switches between the two side-chain conformations. High-resolution monomeric structure of AqpZ, determined by CS-Rosetta calculations using experimentally measured distance restraints related to the R189 side chain, reveals that this side chain is in an open conformation, which is further verified by its water accessibility. All the solid-state NMR experimental results, combining with water permeability essay, suggest a permanently open conformation of the R189 side chain in the synthetic bilayer and native membranes. This study provides new structural insights into the gating mechanism of aquaporins and highlights the significance of lipid bilayer environments in elucidating the molecular mechanism of membrane proteins.

show abstract

Section: ■ Experiments and Methodsmentioning

confidence: 99%

Section: ■ Experiments and Methodsmentioning

confidence: 99%

Section: Experiments and Methodsmentioning

confidence: 99%

Section: Experiments and Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Gating Mechanism of Aquaporin Z in Synthetic Bilayers and Native Membranes Revealed by Solid-State NMR Spectroscopy

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…The E. coli BL21 (DE3) that harboring recombinant AqpZ1 AMS3 was induced with 0.5 mM of IPTG when the optical density (OD) of the cells reached 0.5. Then, after each 4 h interval (4,8,12,16,20, and 24 h) the bacterial cells were harvested and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted.…”

Section: Effect Of Post Induction Timementioning

confidence: 99%

Expression and functional analysis of a recombinant aquaporin Z from Antarctic Pseudomonas sp. AMS3

Balakrishnan,

Rahman,

Noor

et al. 2024

Proteins

View full text Add to dashboard Cite

Aquaporin (AQP) is a water channel protein from the family of transmembrane proteins which facilitates the movement of water across the cell membrane. It is ubiquitous in nature, however the understanding of the water transport mechanism, especially for AQPs in microbes adapted to low temperatures, remains limited. AQP also has been recognized for its ability to be used for water filtration, but knowledge of the biochemical features necessary for its potential applications in industrial processes has been lacking. Therefore, this research was conducted to express, extract, solubilize, purify, and study the functional adaptations of the aquaporin Z family from Pseudomonas sp. AMS3 via molecular approaches. In this study, AqpZ1 AMS3 was successfully subcloned and expressed in E. coli BL21 (DE3) as a recombinant protein. The AqpZ1 AMS3 gene was expressed under optimized conditions and the best optimized condition for the AQP was in 0.5 mM IPTG incubated at 25°C for 20 h induction time. A zwitterionic mild detergent [(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate was the suitable surfactant for the protein solubilization. The protein was then purified via affinity chromatography. Liposome and proteoliposome was reconstituted to determine the particle size using dynamic light scattering. This information obtained from this psychrophilic AQP identified provides new insights into the structural adaptation of this protein at low temperatures and could be useful for low temperature application and molecular engineering purposes in the future.

show abstract

Zika virus NS4B protein targets TANK-binding kinase 1 and inhibits type I interferon production

Sarratea,

Alberti,

Redolfi

et al. 2023

Biochimica et Biophysica Acta (BBA) - General Subjects

View full text Add to dashboard Cite

One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent

Cited by 7 publications

References 32 publications

Gating Mechanism of Aquaporin Z in Synthetic Bilayers and Native Membranes Revealed by Solid-State NMR Spectroscopy

Gating Mechanism of Aquaporin Z in Synthetic Bilayers and Native Membranes Revealed by Solid-State NMR Spectroscopy

Expression and functional analysis of a recombinant aquaporin Z from Antarctic Pseudomonas sp. AMS3

Zika virus NS4B protein targets TANK-binding kinase 1 and inhibits type I interferon production

Contact Info

Product

Resources

About