Jarecki BW , Makino S , Beebe ET , Fox BG , Chanda B .

5T32HL007936-09 NHLBI NIH HHS , GM084140 NIGMS NIH HHS , U54 GM094584-01 NIGMS NIH HHS , T32 HL007936 NHLBI NIH HHS , R01 NS081293 NINDS NIH HHS , R01 GM084140 NIGMS NIH HHS , U54 GM094584 NIGMS NIH HHS

	Figure 1. Cartoon workflow diagram of cell-free protein production and expression in Xenopus laevis oocytes illustrated in two phases.Phase 1: Unique 5′ and 3′ restriction sites appended onto a target gene open-reading frame are used to digest and subclone into a specialized plasmid (pEU-HSBC) for cell-free synthesis. The plasmid DNA is used as a template for in vitro transcription of RNA. Transcribed messenger RNA directs protein translation in wheat-germ cell-free extract supplemented with liposomes to make proteoliposomes. Since the Shaker channels can be recovered from the pellet fraction, the supernatant is discarded and the proteoliposomes are collected after centrifugation. Phase 2: Samples are reconstituted to specified concentrations in a salt buffer and injected into single Xenopus laevis oocytes at the vegetal equator. Currents are measured under voltage-clamp within 24†h.
	Figure 2. Voltage-dependent response and pharmacology of injected samples produced from cell-free translation.(a) SDS-PAGE analysis of proteins produced by wheat-germ cell-free translation. Lanes 1 to 4, representative gel slices from different experiments displaying, from left to right, the molecular weight marker (in kDa), GFP-cytb5t (32†kDa), wildtype and M356C zh4IR channel monomers (71†kDa). The protein samples shown in the gel correspond to those used in other experiments shown in this figure. The family of current traces elicited in response to depolarizing voltage steps (−80 to +60†mV, Δ20†mV) for uninjected oocytes and oocytes injected with GFP-cytb5t (at 0.86 pmol) (b) were negligible compared to the wildtype channel (c) and the M356C channel (d) injected at 0.19 pmol. Current-voltage (I–V) relationship for wildtype (e) and mutant (f) Shaker channels. Inhibition of wildtype (g) and mutant (h) channels in the absence (black) and presence (red) of 10†nM of the pore blocking compound AgTx-2.
	Figure 3. Timecourse and concentration-dependence of the appearance of zh4IR current in protein-injected samples.(a) Maximal K+ current elicited at +60†mV averaged at varying times post-injection for wildtype (Δ4Cys) proteins. Representative families of currents (color-coded relative to the recording time) are displayed above the bars, with a 2†μA scale bar. (b) Average timecourse of expression followed for the same oocyte over a period of 24†h post-injection (n = 4) with data points line-connected for trend comparison. (c) Comparison of maximal current elicited at +60†mV as a function of moles of tetrameric protein injected for four different injection sets. (d) Estimated incorporation efficiency (see Methods) for injection sets displayed in (c). Individual data points are shown in (c–d) to highlight the variability.
	Figure 4. Membrane localization of GFP fused proteins injected into Xenopus laevis oocytes.Grayscale confocal images of (a) GFP-cytb5t (at 2.77 pmol) and (b) zh4IR-GFP (at 0.58 pmol) protein localization within the oocyte membrane at 12†h after proteoliposome injections. Image intensity of the zh4IR-injected oocytes is increased 2-fold for ease of visualization. Arrows indicate the site of injection. Magnified membrane sections taken from the region within the dotted rectangle are displayed below. Scale bar = 200†μm (a, b) and 50†μm (magnified regions, c, d). Corresponding membrane photocurrents (nA) were measured from GFP-cytb5t (e) and zh4IR-GFP (f) at multiple times post-injection.

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