Journal: PLoS ONE

Article Title: An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

doi: 10.1371/journal.pone.0011694

Figure Lengend Snippet: (A) Crystal structure of HA protein (PDB entry 1RUY) showing HA1 (light grey), HA2 (dark grey), and the globular domain of HA 63–286 -RBD (red) used for these studies. (B) Schematic for the construction of HA 63–286 -RBD. The cDNA sequence encoding residues 63–286 of influenza A H1N1 virus (without transmembrane regions) was cloned for expression in Escherichia coli . (C) Schematic representation of the HA 63–286 -RBD containing an N-terminal 6×Histidine tag and an enterokinase cleavage sequence (EkCS). (D) Same as (C) except that this construct contains a periplasmic signal sequence. (E) Amino acid sequence of the N-terminus in both (C) and (D). * indicates the enterokinase cleavage site.

Article Snippet: This simple purification scheme produced HA 63–286 -RBD solutions in the range of 400 to 650 mg/L with purities exceeding 99.5%, as estimated by microelectrophoresis using an Experion® platform from Bio-rad (Hercules, CA).

Techniques: Sequencing, Clone Assay, Expressing, Construct

Journal: PLoS ONE

Article Title: An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

doi: 10.1371/journal.pone.0011694

Figure Lengend Snippet: (A) Hydrophobic (red) and hydrophilic regions (blue) at the surface of protein HA 63–286 -RBD calculated by simulations; (B) The hydrophobicity map of the HA1 subunit expressed by Chiu et al (10) is presented for comparison. (C) Simulation results show that protein HA 63–286 -RBD preserves the conformational antigenic sites Sa, Sb, Ca1, Ca2, Cb computationally predicted by Igarashi et al. for the HA of the influenza A H1N1/CA2009 virus. Three dimensional structures were obtained using Swiss-model. The full length HA of the Influenza A H1N1/1918 virus was taken as a template for the estimation of the most probable structure of protein HA 63–286 -RBD. Visualization and highlighting of immunogenic sites was done using UCSF-Chimera. The structure of the antigenic epitopes of the HA of the influenza A H1N1/CA2009 virus was taken from Igarashi et al. . They are also consistent with structural data published recently by Xu et al .

Article Snippet: This simple purification scheme produced HA 63–286 -RBD solutions in the range of 400 to 650 mg/L with purities exceeding 99.5%, as estimated by microelectrophoresis using an Experion® platform from Bio-rad (Hercules, CA).

Techniques:

Journal: PLoS ONE

Article Title: An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

doi: 10.1371/journal.pone.0011694

Figure Lengend Snippet: (A) Protein profile of cell lysates from culture experiments of E. coli C41, BL21 (DE3) pLysS or Rosetta-gami transformed with genes to produce (1) GFP+histidine tag (clone C41 1); (2) GFP+histidine tag (clone C41 2); (3) GFP+histidine tag (clone C41 3); (4) negative control, C41(5) HA 63–286 -RBD (clone C41 1); (6) HA 63–286 -RBD (clone C41 2); (7) HA 63–286 -RBD (clone Rosetta-gami clone 1); (8) HA 63–286 -RBD (clone Rosetta-gami clone 2). (9) Precision Plus Kaleidoscope molecular mass ruler showing 25 kD (pink) and 20 kD (blue) bands. The blue arrow indicates the 26 kD band corresponding to HA 63–286 -RBD. (B) SDS-PAGE showing (1) the soluble and (2) insoluble fraction of the C41 strain lysate after 8 hours induction with 1mM IPTG. (C) SDS-PAGE showing the protein profiles at different stages of recovery, purification and on-column refolding. (1) Crude lysate of the 8M urea solubilized inclusion bodies, (2) Unbound fraction, (3) 1st wash step, (4) 2nd wash step, (5,6) refolding steps, (7–12) Elution fraction using imidazole 300 mM, (13) chromatographic resin. (M) Precision Plus Kaleidoscope molecular mass ruler.

Article Snippet: This simple purification scheme produced HA 63–286 -RBD solutions in the range of 400 to 650 mg/L with purities exceeding 99.5%, as estimated by microelectrophoresis using an Experion® platform from Bio-rad (Hercules, CA).

Techniques: Transformation Assay, Negative Control, SDS Page, Purification

Journal: PLoS ONE

Article Title: An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

doi: 10.1371/journal.pone.0011694

Figure Lengend Snippet: (A) Bars 1–8, in gray tones, correspond to absorbance signals from non-exposed subjects (samples taken from March to May 2008). Bar 9, in black, shows the average absorbance value of samples 1 to 8. Bars 10 to 14, shown in colour, correspond to absorbance signals from Influenza A/H1N1 negative subjects. Bars 15–23, shown in colour, correspond to absorbance signals from samples of Influenza A H1N1 positive subjects (diagnosed two or three weeks previously by RT-PCR). Error bars represent one standard deviation (B) Proper refolding (biorecognition of antibodies from a positive patient), was evaluated for 4 different production batches of HA 63–286 -RBD. Batch 5 is a reference batch where HA 63–286 -RBD was expressed in its soluble form using a signal peptide for periplasmic expression.

Article Snippet: This simple purification scheme produced HA 63–286 -RBD solutions in the range of 400 to 650 mg/L with purities exceeding 99.5%, as estimated by microelectrophoresis using an Experion® platform from Bio-rad (Hercules, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Expressing

Journal: PLoS ONE

Article Title: An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

doi: 10.1371/journal.pone.0011694

Figure Lengend Snippet: (A) Evolution of specific immune response of ferret 4A to intramuscular application of HA 63–286 -RBD. Arrows indicate the days at which dosages of the protein were administered. (B) Evolution of a specific immune response of ferret 4C to sub-dermal application of HA 63–286 -RBD. Arrows indicate the days at which dosages of the protein were administered. Error bars indicate one standard deviation for three replicates in an ELISA experiment. Specific immune response is expressed in absorbance units (absorbance signal in the assay with ferret serum minus absorbance in negative control). (C and D) Effect of adjuvant on immune response and comparison of immunogenic activity of different doses of HA 63–286 -RBD administered to ferrets. Specific immune response is expressed in absorbance units (absorbance signal in the assay with ferret serum minus absorbance in negative control) at days 0 (gray), 3 (yellow), 15 (green), and 20 (blue) after the first vaccination dose. Error bars indicate one standard deviation with respect to the mean value for replicates of the experiment in different ferrets. Arrows indicate days of application of a second dose (if administered).

Article Snippet: This simple purification scheme produced HA 63–286 -RBD solutions in the range of 400 to 650 mg/L with purities exceeding 99.5%, as estimated by microelectrophoresis using an Experion® platform from Bio-rad (Hercules, CA).

Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay, Negative Control, Activity Assay

Journal: PLoS ONE

Article Title: An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

doi: 10.1371/journal.pone.0011694

Figure Lengend Snippet: (A) Averages from four different symptom indexes are compared for vaccinated (green bars) and non-vaccinated ferrets (red bars). Error bars represent one standard deviation. (B) Averages of the overall sickness index (as defined in text) are compared for vaccinated (green bars) and non-vaccinated ferrets (red bars). Error bars represent one standard deviation. (C) Evolution of body weight loss in ferrets challenged intra-nasally with infective dosages of influenza A/H1N1/2009 virus (at day 0). Averages of daily Δweight (Weight dayx –Weight basal ) for five non-vaccinated controls (▪), five ferrets administered with a single dose of 125µg of HA 63–286 -RBD (○); three ferrets administered with a single dose of 200 µg of HA 63–286 -RBD (•); three ferrets administered with a single dose of 275 µg of HA 63–286 -RBD (♦); and all sixteen vaccinated ferrets (□) are compared. Error bars represent one standard deviation. (D) Evolution of body temperature in ferrets challenged intra-nasally with infective dosages of Influenza A/H1N1/2009 virus (at day 0). Averages of daily ΔT (T dayx –T basal ) for five non-vaccinated controls (▪), five ferrets administered with a single dose of 275 µg of HA 63–286 -RBD in a double dose (▴), and 275 µg of HA 63–286 -RBD in a single dose are compared (♦). Error bars represent one standard deviation.

Article Snippet: This simple purification scheme produced HA 63–286 -RBD solutions in the range of 400 to 650 mg/L with purities exceeding 99.5%, as estimated by microelectrophoresis using an Experion® platform from Bio-rad (Hercules, CA).

Techniques: Standard Deviation

Journal:

Article Title: SynGAP regulates steady state and activity-dependent phosphorylation of cofilin

doi: 10.1523/JNEUROSCI.4695-08.2008

Figure Lengend Snippet: Steady-state activity of PAK and cofilin is altered and regulation cofilin activity by the NMDA receptor is deranged in synGAP mutant hippocampal cultures. Wild type, synGAP+/-, and synGAP-/- hippocampal cultures (DIV 15-18) were treated with 25 μM NMDA for 15 sec - 5 min and then immunoblotted with anti-phospho-Thr423 PAK(1-3) or anti-phospho-Ser3 cofilin antibody. Phosphoprotein levels were measured and quantified as described under methods. A, The levels of phospho-PAK and phospho-cofilin were basally elevated in untreated synGAP-/- hippocampal cultures. Changes in phosphorylation of PAK and cofilin are expressed as a percentage of the wild type untreated control values (synGAP+/- phospho-PAK = 153 ± 29.2% of wt, n = 7 litters; synGAP-/- phospho-PAK* = 202 ± 43.4% of wt, P = 0.006, n = 5 litters; synGAP+/- phospho-cofilin = 121 ± 18.6% of wt, n = 11 litters; synGAP-/- phopho-cofilin* =141 ± 12.9% of wt, P = 0.012, n = 10 litters). B, Despite the basal increase in phosphorylated PAK in the mutants, treatment with NMDA still induces an additional increase in phospho-PAK, similar to that in wild types, when each genotype is normalized to its own untreated genotype control (wild type; 15 sec = 267 ± 46.4%, 1 min = 294 ± 54.7%, 3 min = 286 ± 63.7%, 5 min = 132 ± 67.5% of wild type control; N = 8 litters) (SynGAP+/-; 15 sec = 316 ± 58.5%, 1 min = 315 ± 58.4%, 3 min = 256 ± 45.7%, 5 min = 134 ± 40.6% of synGAP+/- control; N = 8 litters) (SynGAP-/-; 15 sec = 252 ± 40.1%; 1 min = 415 ± 130%; 3 min = 322 ± 95.5%; 5 min = 224 ± 75.7% of synGAP-/- control; N = 8 litters). C, NMDA induces significantly less dephosphorylation of cofilin in synGAP mutant compared to wild-type cultures (wild type: 15 sec = 59.7 ± 7.37%; 1 min = 67.4 ± 7.05%; 3 min = 68.7 ± 6.68%; 5 min = 73.4 ± 78.91% of wild type control; N = 6 litters) (SynGAP+/-: 15 sec = 83.5 ± 5.64%; 1 min = 77.8 ± 5.29%; 3 min = 77.2 ± 7.04%; 5 min = 80.4 ± 5.16% of SynGAP+/- control; N = 4 litters) (SynGAP-/-: 15 sec* = 96.9 ± 1.07%, P = 0.006; 1 min* = 94.6 ± 4.87%, P = 0.37; 3 min = 81.0 ± 7.48%; 5 min = 78.0 ± 5.85% of SynGAP-/- control; N = 5 litters; one-way ANOVA comparing the three genotypes at each time point, followed by Tukey-Kramer multiple comparison test). All time points were significantly less than 100% in the wild type neurons; the 1, 3, and 5 min time points were significantly less that 100% in the synGAP+/- neurons; and only the 5 min time point was significantly less than 100% in the synGAP-/- neurons (P < 0.05, 2-tailed, one-sample t-test). Example blots are shown on right. Graphs represent geometric means ± SEGM.

Article Snippet: B , Despite the basal increase in phosphorylated PAK in the mutants, treatment with NMDA still induces an additional increase in phospho-PAK, similar to that in wild types, when each genotype is normalized to its own untreated genotype control (wild type; 15 sec = 267 ± 46.4%, 1 min = 294 ± 54.7%, 3 min = 286 ± 63.7%, 5 min = 132 ± 67.5% of wild type control; N = 8 litters) ( SynGAP +/- ; 15 sec = 316 ± 58.5%, 1 min = 315 ± 58.4%, 3 min = 256 ± 45.7%, 5 min = 134 ± 40.6% of synGAP +/- control; N = 8 litters) ( SynGAP -/- ; 15 sec = 252 ± 40.1%; 1 min = 415 ± 130%; 3 min = 322 ± 95.5%; 5 min = 224 ± 75.7% of synGAP -/- control; N = 8 litters).

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, Control, De-Phosphorylation Assay, Comparison