Fig 1.
The coding sequence of SLC46A1 gene, nucleotides from 99 to 1478, together with two recognition sites for BamHI and EcoRI restriction enzymes, were cloned into pEXP5-NT/TOPO vector purchased from ThermoFisher Scientific. The correct DNA sequence of the PCFT construct was confirmed by Genewiz.
Fig 2.
Schematic for cell-free expression of PCFT in the presence of nanodiscs.
T7 S30 Extract, Circular, and S30 Premix Plus are components of the S30 T7 High-Yield Protein Expression System purchased from Promega. The pEXP5-PCFT plasmid is the PCFT construct shown in Fig 1. Empty LPNs containing POPC, DMPG, or DMPC were added to a cell-free reaction mixture. Soluble PCFT was observed when PCFT was cell-free expressed in the presence of the nanodisc.
Fig 3.
Cell-free expression of Proton-Coupled Folate Transporter (PCFT).
The arrows indicate the position of monomeric PCFT (~37 kDa). A) Cell-free expression of PCFT in the absence of LPNs. The “Control” lane reactions did not contain PCFT-pEXP5-NT plasmid. β-mercaptoethanol (BME) was added to some reactions as indicated. B) Cell-free expression of PCFT in the presence of 20 μM or 40 μM LPNs containing POPC, DMPG, or DMPC lipids. The “PCFT” lane reactions did not contain any LPNs.
Fig 4.
Solubilization of PCFT in different lipid concentrations and compositions.
A) Cell-free expression of PCFT in the presence of 20 μM, 40 μM, or 80 μM LPNs containing POPC, DMPG, or DMPC lipid. PCFT lane did not contain any LPNs. The PCFT arrow indicates PCFT protein bands, and the MSP arrow indicates membrane scaffold protein (MSP) of LPNs. B) PCFT solubilization in 20 μM, 40 μM, or 80 μM LPNs containing POPC, DMPG, or DMPC lipid. The PCFT band intensity was quantified in western blots of supernatant fractions and normalized to the PCFT band derived from 80 μM DMPC LPNs. The data show the mean ± SEM from n = 3 independent experiments. The highest level of solubilized PCFT was found in LPNs containing DMPC lipid. LPNs containing POPC lipid yielded minimal levels of solubilized PCFT. Statistical significance was determined using was determined using one-way ANOVA with Tukey’s multiple-comparisons test in Prism 6 Software (GraphPad Prism). Significance is indicated vs “PCFT” sample without LPNs as ** p ≤ 0.01, *** p ≤ 0.001, and p ≤ ****0.0001). Nonsignificant p value is shown as ns.