A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system over- comes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (\u3e1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli

Antibody-Mediated Targeting of Iron Oxide Nanoparticles to the Folate Receptor Alpha Increases Tumor Cell Association In Vitro and In Vivo

Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy

Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method

<i>In vitro</i> binding affinity of Tfab and Tmab.

<p>*IC₅₀ values reflect concentration of Tfab required to compete 50% of saturating Tmab IgG.</p><p>Errors are standard deviations from technical triplicates.</p><p><i>In vitro</i> binding affinity of Tfab and Tmab.</p

TEM imaging of subcellular localization following <i>in vitro</i> binding of IONP-Tfab to HER2+ breast cancer cells.

<p>(<b>A</b>) At 20,000X magnification in SKBR3 cells, 30 nm IONP-Tfab localize primary to intracellular vesicles with a smaller proportion remaining bound to the cell surface (arrows). (<b>B</b>) At 20,000X magnification in BT-474 cells, 30 nm IONP-Tfab exhibit similar localization. (<b>C</b>) At 10,000X magnification in SKBR3 cells, 100 nm IONP-Tfab are mainly found in intracellular vesicles (arrows). (<b>D</b>) At 20,000X magnification in BT-474 cells, 100 nm IONP-Tfab exhibit similar localization. Scale bars are 100 nm (A, B, D) and 500 nm (C).</p