Journal: PLoS ONE

Article Title: Synthesis of UDP-apiose in Bacteria: The marine phototroph Geminicoccus roseus and the plant pathogen Xanthomonas pisi

doi: 10.1371/journal.pone.0184953

Figure Lengend Snippet: Analysis of in microbe nucleotide sugars by HILIC-LC-ESI-MS/MS. (A) top panel elution of standard (Std): UDP-GlcA, UDP-Xyl and UDP-arabinopyranose (UDP-Ara p ); Nucleotide sugars were extracted from E . coli cells induced to express genes encoding CeUAS, GrUAS, XpUAS or empty vector as the control (bottom panel). [M-H] - ions diagnostic for UDP-pentose ( m/z 535.0, solid line), UDP-hexuronic acid ( m/z 579.0, dashed line) and Park’s nucleotide ( m/z 595.6, dotted line) are displayed. Park’s nucleotide is a UDP-MurNAc-pentapeptide that is used as an internal standard for nucleotide-sugar detection as it is abundantly made in E . coli . The m/z signal for CeUAS and XpUAS is amplified by a factor of 10. (B) Second stage MS fragmentation data for the peaks at the indicated retention times; Left column 11.0 min and right column 12.0 min. MS/MS ions at m/z 323.0, 211.0, 403.0 are consistent with predicted fragmentation of a UDP-sugar into [UMP-H] - , [Ura-2H] - , and [UDP-H 2 O-H] - , respectively.

Article Snippet: Because no axenic monoculture of C . entotheonella was available, a synthetic ORF gene corresponding to the nucleotide sequence of CeUAS was obtained (GenScript, Piscataway, NJ, USA).

Techniques: Hydrophilic Interaction Liquid Chromatography, Tandem Mass Spectroscopy, Plasmid Preparation, Control, Diagnostic Assay, Amplification

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells

doi: 10.3389/fbioe.2022.908509

Figure Lengend Snippet: Production of PfRipr5 at 2 L stirred-tank bioreactor (STB) scale. (A) Relative PfRipr5 concentration at the TOH of each cell line. (B) SDS-PAGE of purified PfRipr5. (C) Size distribution profile of purified PfRipr5 assessed by dynamic light scattering. (D) ELISA of purified PfRipr5 using an anti- P. falciparum PfRipr mouse monoclonal antibody (mAb) 29B11. For figure (A) : relative PfRipr5 expression was assessed by a densitometry analysis of western blot as described in the M&M section. For figure (B) : L denotes pre-stained protein standard SeeBlue® Plus2, R denotes reduced sample, NR denotes non-reduced sample. Data are relative to one biological replicate ( n = 1).

Article Snippet: PfRipr5 nucleotide sequence ( ) was codon-optimized for expression in insect or human cells, with an N-terminal gp67 SP sequence to allow secretion into the culture medium, a C-terminal His 6 -tag to allow purification, and a Kozak consensus sequence (GCCACT or ACC for human or insect expression plasmid, respectively) as the translation initiation site (synthetized by GenScript, Leiden, the Netherlands).

Techniques: Concentration Assay, SDS Page, Purification, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells

doi: 10.3389/fbioe.2022.908509

Figure Lengend Snippet: Optimization of PfRipr5 production using insect cells. (A) Optimization strategies devised. (B) Relative PfRipr5 concentration at the TOH between each optimization condition and the baseline production setup (infection with rBAC gp67 without culture temperature shift). A denotes alanine, G denotes glycine, and S denotes serine. Infections were performed using CCI = 2 × 10 6 cell/mL and MOI = 0.1 pfu/cell. Data are expressed as mean ± standard deviation and is relative to three biological replicates ( n = 3).

Article Snippet: PfRipr5 nucleotide sequence ( ) was codon-optimized for expression in insect or human cells, with an N-terminal gp67 SP sequence to allow secretion into the culture medium, a C-terminal His 6 -tag to allow purification, and a Kozak consensus sequence (GCCACT or ACC for human or insect expression plasmid, respectively) as the translation initiation site (synthetized by GenScript, Leiden, the Netherlands).

Techniques: Concentration Assay, Infection, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells

doi: 10.3389/fbioe.2022.908509

Figure Lengend Snippet: Production of the PfRipr5 recombinant protein. (A) Kinetics of cell growth and viability upon infection of insect High Five (green) and Sf 9 (orange) cells at different combinations of cell concentration at infection (CCI) and multiplicity of infection (MOI). (B) Kinetics of cell growth and viability upon transfection of human HEK293 cells at different combinations of the PfRipr5 plasSmid DNA (pDNA) concentration (pDNA) and ratio pDNA:PEI. (C) Relative PfRipr5 concentration at the time-of-harvest (TOH) of each production condition. Data are expressed as mean ± standard deviation. For insect cells, data are relative to three biological replicates ( n = 3). For human cells, data are relative to one biological replicate ( n = 1). For figure (A,B) , VCC denotes viable cell concentration. For figure (C) , relative PfRipr5 expression was assessed by a densitometry analysis of western blot as described in the M&M section; graph is normalized at 1 for the best condition using human cells [i.e., (pDNA) = 0.5 mg/L and ratio pDNA:PEI = 1:2 (w:w)].

Article Snippet: PfRipr5 nucleotide sequence ( ) was codon-optimized for expression in insect or human cells, with an N-terminal gp67 SP sequence to allow secretion into the culture medium, a C-terminal His 6 -tag to allow purification, and a Kozak consensus sequence (GCCACT or ACC for human or insect expression plasmid, respectively) as the translation initiation site (synthetized by GenScript, Leiden, the Netherlands).

Techniques: Recombinant, Infection, Concentration Assay, Transfection, Standard Deviation, Expressing, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells

doi: 10.3389/fbioe.2022.908509

Figure Lengend Snippet: Production of PfRipr5 at 2 L STB scale using the optimized production strategy (infection of insect High Five cells with rBAC A-GGSGG and culture temperature shift from 27 to 22°C at TOI). (A) Kinetics of cell growth and viability throughout infection. (B) Western blot identification of PfRipr5 in bulk and purified samples. (C) Production yield following purification. (D) SDS-PAGE of purified PfRipr5. (E) Size distribution profile of purified PfRipr5 assessed by dynamic light scattering. (F) ELISA of purified PfRipr5 using an anti- P. falciparum PfRipr mouse mAb 29B11. For figure (A) , VCC denotes viable cell concentration. For figure (B) , DPI denotes day post-infection, (+) denotes the positive control (PfRipr5 produced by WGCFS). For figure (B) and figure (D) , L denotes pre-stained protein standard SeeBlue® Plus2, R denotes reduced sample, and NR denotes non-reduced sample. Infection was performed using CCI = 2 × 10 6 cell/mL and MOI = 0.1 pfu/cell. Data are relative to one biological replicate ( n = 1).

Article Snippet: PfRipr5 nucleotide sequence ( ) was codon-optimized for expression in insect or human cells, with an N-terminal gp67 SP sequence to allow secretion into the culture medium, a C-terminal His 6 -tag to allow purification, and a Kozak consensus sequence (GCCACT or ACC for human or insect expression plasmid, respectively) as the translation initiation site (synthetized by GenScript, Leiden, the Netherlands).

Techniques: Infection, Western Blot, Purification, SDS Page, Enzyme-linked Immunosorbent Assay, Concentration Assay, Positive Control, Produced, Staining