Purification and Reconstitution of PaNhaP

The synthetic gene for wild-type PaNhaP (PaNhaP_WT) was cloned with a C-terminal cysteine protease domain fusion into the pET21a plasmid. The PaNhaP mutation Glu-73 to Ala (PaNhaP_E73A) was introduced by site-directed mutagenesis (19 (link)). The resulting plasmids were used to transform E. coli C41-(DE3) cells and target proteins were purified as described (5 (link)). Purified proteins were reconstituted at a lipid to protein ratio (w/w) of 4 into liposomes prepared from E. coli polar lipids (Avanti Polar Lipids, Inc., Alabaster, AL) as described (5 (link)).

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Călinescu O., Linder M., Wöhlert D., Yildiz Ö., Kühlbrandt W, & Fendler K. (2016). Electrogenic Cation Binding in the Electroneutral Na+/H+ Antiporter of Pyrococcus abyssi. The Journal of Biological Chemistry, 291(52), 26786-26793.

Publication 2016

E. coli polar lipids

Ala glu Alabaster Cells Cysteine protease E coli Glu ala Lipid a Lipids Liposomes Mutation Plasmids Protein Site directed mutagenesis Synthetic gene

Corresponding Organization :

Other organizations : Carol Davila University of Medicine and Pharmacy, Max Planck Institute of Biophysics

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Variable analysis

independent variables

PaNhaP mutation Glu-73 to Ala (PaNhaP_E73A)

dependent variables

Purified proteins were reconstituted at a lipid to protein ratio (w/w) of 4 into liposomes

control variables

Wild-type PaNhaP (PaNhaP_WT) was cloned with a C-terminal cysteine protease domain fusion into the pET21a plasmid
Resulting plasmids were used to transform E. coli C41-(DE3) cells
Target proteins were purified as described (5 (link))
Purified proteins were reconstituted into liposomes prepared from E. coli polar lipids (Avanti Polar Lipids, Inc., Alabaster, AL) as described (5 (link))

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