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  1. NiCo21(DE3): A BL21(DE3) Derivative Designed for Expression and Purification of His-tagged Recombinant Protein (2015)

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly co-eluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA and optionally AceE fused at their C-terminus to a chitin binding domain (CBD), and the protein GlmS with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flow-through step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

  2. One Ring To Bind Them All: Is HemeBiosynthesis A Factor In Wolbachia-Filarial Nematode Endosymbiosis? (2015)

    Transmitted by insect vectors, human nematode-based filariasis causes debilitating diseases affecting nearly 150 million people with 1.2 billion individuals at risk in 80 countries. Genomic sequencing has revealed that many human filarial nematodes, such as Wuchereria bancrofti, Brugia malayi and B. timori, (causative agents of lymphatic filariasis (LF)) and Onchocerca volvulus (causative agent of onchocerciasis (river blindness)) contain the obligate endosymbiont, Wolbachia. Genome sequencing of B. malayi (Bm) and its Wolbachia (wBm) identified a number of metabolites implicated in the host-endosymbiont interaction, one of which was heme, a co-factor in a number of enzymes and essential to many biological processes. Although the Bm genome encodes a functional ferrochelatase gene (the final step in the heme biosynthetic pathway and a product of lateral gene transfer), like other nematodes, it is incapable of synthesizing heme. However, the wBm genome contains a functional heme synthesis pathway, leading to the hypothesis that wBm may supply Bm with heme

  3. Molecular Diagnostics for Gastrointestinal Parasites and Impact on Intestinal Microbiota in Rural Agentinian Children (2015)

    • 2 billion GI parasite infections worldwide
      • Poorest and resource-deprived communities
    • Standard method of diagnosis: Stool microscopy
      • Sensitivity variable depending on prevalence, species, and concentration method
        • 50-90% sensitivity
      • Underestimates polyparasitism
    • qPCR is rapid, quantitative, and high throughput species-specific method
    • GI parasites may disrupt normal intestinal microbiota
      • Decreased biodiversity is associated with disease
        • Malabsorption
        • Inflammatory bowel diseases

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