exp2 protein  (GenScript corporation)

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) IFA images of condΔEXP1 schizont stages expressing EXP1 wt-Ty probed with α-HA and α-Ty show localization of EXP1*-HA and EXP1 wt-Ty in the PVM. α-MSP1 (MSP1) labels the PPM. DAPI, nuclei; scale bars, 5 μm. Right: immunoblot of extracts of cell line on the left probed with α-Ty to detect EXP1wt-Ty and α-SBP1 as control for a TM protein. Saponin was used to separate the parasite pellet (“P”) from the supernatant (“SN”) containing PV and host cell content. See S3 Fig for IFAs and immunoblots of all complementation constructs. (B) Relative activity of the EXP1 complementation constructs indicated. Except where otherwise indicated, constructs were expressed under the nmd3 (mid) promoter. The complementation capacity of every tested construct was calculated as relative activity to the EXP1wt-Ty construct under the nmd3 promoter, which was set as 100% (right dotted green line). ΔEXP1 was set to 0% (left dotted green line). Each data point (red dot) shows growth of rapalog-treated versus unexcised parasites at the end of a 5-day growth assay relative to the growth of the wt construct; n ≥ 4 independent experiments per cell line. Error bars: SD. See for activity of all complementation constructs. (C) Immunoblot of lysates of condΔEXP1 parasites expressing EXP1wt low , EXP1 mid , and EXP1 high probed with α-Ty (EXP1wt-Ty), α-HA (EXP1*-HA), and α-BIP (loading control). (D) Densitometric analysis of EXP1wt expression levels (C) under low, mid, and high promoters relative to the mid promoter (green). Mean of 3 independent experiments. Error bars: SD. (E) Immunoblot of extracts of +/−formaldehyde (PFA) treated cell lines expressing the indicated constructs probed with α-Ty. Single asterisk: monomer; double asterisk: dimer. BIP, binding immunoglobulin protein; DIC, differential interference contrast; EXP1, exported protein 1; HA, triple hemagglutinin tag; IFA, immunofluorescence assay; MSP1, merozoite surface protein 1; PPM, parasite plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuolar membrane; SBP1, skeleton binding protein 1; TM, transmembrane; wt, wild type.

Article Snippet: To obtain EXP2-GFP expressed under the nmd3 promoter, EXP2 was synthesized with a different codon usage (GenScript) and PCR amplified with overhanging sequences to fuse it with GFP and clone it into p1xNLS-FRB-mCherry nmd3 digested with XhoI and XmaI using Gibson cloning.

Techniques: Expressing, Western Blot, Control, Construct, Activity Assay, Growth Assay, Binding Assay, Immunofluorescence, Clinical Proteomics, Membrane

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Schematic of the region of EXP1 with the proposed catalytic site of the GST activity and of the mutations introduced. (B) Relative activity of the EXP1 complementation constructs indicated. Green lines: activity of EXP1wt ( nmd3 , mid) (set as 100%) and absence of activity (ΔEXP1) set as 0%; n ≥ 4 independent experiments per cell line. (C) Live cell images of condΔEXP1 parasites incubated with CM-H 2 DCFDA. Scale bars: 5 μm. (D) Fluorescence intensity of matching stages of control and ΔEXP1 parasites (rapalog) incubated with CM-H 2 DCFDA (C). Results from 3 independent experiments with a total of n = 55 control and n = 53 ΔEXP1 cells. Green line, mean; error bars, SD. (E) FC growth curves of synchronous condΔEXP1 parasites and the complementation cell line EXP1 wt low after one cycle with (red) and without (black) rapalog (see ) grown in RPMI alone or supplemented with the compounds indicated. One representative of n = 3 independent biological replicas. (F) Left, growth curves of EXP1wt low parasites ± E64 starting after one growth cycle ± rapalog (see ). Right, Giemsa smears of parasites on day 2 (after incubation with E64) and day 3 (after removal). Arrowheads: swollen food vacuole. One representative of n = 3 biological replicas. (G) Effect of E64 treatment on survival of DHA and rapalog-treated EXP1wt low parasites versus untreated. Mean of n = 3 independent experiments. (H) Left, dose-response curves of the parasite lines indicated treated with DHA (0–50 nM). Right, DHA IC 50 values for these cell lines ± rapalog. Mean of n ≥ 3 experiments. (I) RSAs of the indicated complementation cell lines and a Kelch13 C580Y mutant line. Data points are percent survival of DHA treated versus untreated parasites ± rapalog. Mean of n ≥ 3 per cell line. (D, G, and H), two-tailed unpaired t test; P values indicated; (B, D, and G), error bars, SD. CM-H 2 DCFDA,; DHA, dihydroartemisinin; DIC, differential interference contrast; EXP1, exported protein 1; FC, flow cytometry; GST, glutathione S-transferase; IC 50 , half maximal inhibitory concentration; RPMI, Roswell Park Memorial Institute; RSA, ring-stage survival assay; wt, wild type.

Article Snippet: To obtain EXP2-GFP expressed under the nmd3 promoter, EXP2 was synthesized with a different codon usage (GenScript) and PCR amplified with overhanging sequences to fuse it with GFP and clone it into p1xNLS-FRB-mCherry nmd3 digested with XhoI and XmaI using Gibson cloning.

Techniques: Activity Assay, Construct, Incubation, Fluorescence, Control, Mutagenesis, Two Tailed Test, Flow Cytometry, Concentration Assay, Clonogenic Cell Survival Assay