e. coli codon optimised orf  (GenScript corporation)

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: A) Diagram of the PfEMP1 domain architecture of known rosette-mediating variants [ – ]. The rosetting-associated head structure, DBLα1.5/6/8-CIDRβ/γ/δ, is boxed. Domains that bind erythrocytes (RBC) or serum proteins (IgM and alpha2Macroglobulin, α2M) are indicated. B) Diagram of domain cassettes (DCs) [as described by ] which are relevant to the rosetting PfEMP1 variants characterised previously.

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques:

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: A) IgM binding by KE10R+ infected erythrocytes detected by flow cytometry. Forward and side scatter were used to gate on erythrocytes and exclude debris (left panel) and PfEMP1-expressing infected erythrocytes were detected by staining with 20μg/ml of polyclonal rabbit IgG against NTS-DBLα of KE10VAR_R1 followed by 1/1000 dilution of Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibody and 1/2500 dilution of Vybrant DyeCycle Violet (middle panel Q2). IgM staining of the Q2 cell population was detected with 1/1000 dilution of an Alexa Fluor 488-conjugated goat anti-human IgM heavy chain antibody (red). The negative control (blue) was parasites grown in IgM-depleted medium and stained with the same antibodies. Results are representative of two independent experiments. B) Effect of low-dose trypsinisation on rosetting. Purified infected erythrocytes were treated with 0.5, 1 or 5 μg/ml of trypsin for 5 mins and the rosette frequency relative to a control with no added enzyme was calculated. The mean and standard deviation from three independent experiments per parasite line is shown. The rosette frequency of the untreated control was between 46%-81% (KE10R+), 45%-89% (PC0053R+), 16%-30% (KE11R+) and 61%-91% (KE08R+). C-E) Effect of heparin on rosetting. The mean and standard error from two or three independent experiments per parasite line is shown.

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques: Binding Assay, Infection, Flow Cytometry, Expressing, Staining, Negative Control, Purification, Control, Standard Deviation

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: Fluorescence intensity histograms of rosetting (R+) and non-rosetting (R-) parasite lines stained with rabbit IgG against rosetting PfEMP1 variants (red) or negative control (rabbit IgG against the NTS-DBLα domain of an irrelevant PfEMP1 variant, HB3VAR03, blue). A) parasite line KE10R+ with antibodies to KE10VAR_R1 NTS-DBLα. B) parasite line KE10R- with antibodies to KE10VAR_R1 NTS-DBLα. C) parasite line PC0053R+ with antibodies to PC0053VAR_R1 NTS-DBLα. D) parasite line PC0053R- with antibodies to PC0053VAR_R1 NTS-DBLα. E) parasite line KE08R+ with antibodies to KE08VAR_R1 NTS-DBLα. F) parasite line KE08R- with antibodies to KE08VAR_R1 NTS-DBLα. G) parasite line KE08R+ with antibodies to KE08VAR_R2 NTS-DBLα. H) parasite line KE08R- with antibodies to KE08VAR_R2 NTS-DBLα. I) parasite line KE08R+ with antibodies to KE08VAR_R1 CIDR. J) parasite line KE08R- with antibodies to KE08VAR_R1 CIDR. K) parasite line KE08R+ with antibodies to KE08VAR_R2 CIDR. L) parasite line KE08R- with antibodies to KE08VAR_R2 CIDR. PfEMP1 was detected with 20μg/ml rabbit IgG against PfEMP1 and 1/1000 dilution of Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibody. Gates shows the percentage of mature infected erythrocytes positive for the variant. The rosette frequency at the time of staining was 70–90% for the R+ lines and <2% for the R- lines, and the percentage of positive staining infected erythrocytes (APC-A+, top right corner of each histogram) closely matched the rosette frequency in each parasite line. Results are representative of at least two experiments for each parasite line.

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques: Fluorescence, Staining, Negative Control, Variant Assay, Infection

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: Antibodies to the candidate rosette-mediating PfEMP1 variant NTS-DBLα domains were tested in rosette-disruption assays over a range of concentrations from 0.1–100 μg/mL. The rosette frequency in the presence of antibody is shown as the proportion of the control value with no added antibody. IgG from a non-immunised (NI) rabbit and from a rabbit immunised with the NTS-DBLα domain of a non-rosetting PfEMP1 variant HB3VAR03 were used as negative controls. Data represent three independent experiments with the mean and standard deviation of the three experiments shown. A) parasite line KE10R+; the rosette frequency of the no antibody control ranged from 71%-87%. B) Parasite line PC0053R+; the rosette frequency of the no antibody control ranged from 49%-60%. C) Parasite line KE08R+; the rosette frequency of the no antibody control ranged from 69%-92%. Data were analyzed by two-tailed paired t tests corrected for multiple comparisons with the Holm-Sidak method ** P<0.01, *** P<0.001, **** P<0.0001.

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques: Variant Assay, Disruption, Control, Standard Deviation, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: A) Recombinant proteins were incubated with uninfected erythrocytes and bound protein detected with specific antibodies to each domain. The positive control was the NTS-DBLα domain of the well-characterised rosetting variant IT4VAR60 [ , ] and negative controls were the NTS-DBLα domains from human brain endothelial cell binding PfEMP1 variants HB3VAR03 (PFHB3_130080100), IT4VAR07 (PFIT_060036700), 3D7_PFD0020c (PF3D7_0400400), PC0053-C.g96 and PC0053-C.g410 that are known to be non-rosetting . The mean and standard deviation of the Alexa Fluor 488 median fluorescence intensity from at least three independent experiments per protein is indicated. Data were log transformed and analysed by one way ANOVA with Dunnett’s multiple comparisons test compared to the “Nil” (no added protein) control. **** P<0.0001. B-G) Example Alexa Fluor 488 fluorescence intensity histograms of recombinant PfEMP1 domains bound to erythrocytes and detected by indirect immunofluorescence (red) compared to a non-rosetting NTS-DBLα domain negative control (blue). The domain tested is indicated below each histogram.

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques: Recombinant, Incubation, Positive Control, Variant Assay, Binding Assay, Standard Deviation, Fluorescence, Transformation Assay, Control, Immunofluorescence, Negative Control

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: IgM was coated onto the wells of an ELISA plate and individual PfEMP1 recombinant protein domains were added at four different 5-fold dilutions. Wells with no IgM were included as negative controls. Binding was detected using an HRP-conjugated anti-His tag antibody with TMB substrate and the absorbance read at 450nm. The domains are shown from N- to C-terminal and the panels are colour-coded as previously described . One representative experiment is shown out of at least two performed for each domain.

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: var genes encoding rosetting PfEMP1 variants * .

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques:

Journal: PLOS Pathogens

Article Title: Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

doi: 10.1371/journal.ppat.1012434

Figure Lengend Snippet: var genes encode non-rosetting PfEMP1 variants * .

Article Snippet: Codon optimised sequences for E . coli expression of PfEMP1 domains with a Bam HI site at the 5’ end and an Nhe I site at the 3’ end were obtained from Twist Biosciences (San Francisco, California) and ligated into the expression vector modified pET15b (pET15b conv) [ ].

Techniques: Binding Assay