ABSTRACT
Protein-conjugated magnetic nanoparticles (mNPs) are promising tools for a variety of biomedical applications, from immunoassays and biosensors to theranostics and drug-delivery. In such applications, conjugation of affinity proteins (e.g., antibodies) to the nanoparticle surface many times compromises biological activity and specificity, leading to increased reagent consumption and decreased assay performance. To address this problem, we engineered a biomolecular magnetic separation system that eliminates the need to chemically modify nanoparticles with the capture biomolecules or synthetic polymers of any kind. The system consists of (i) thermoresponsive magnetic iron oxide nanoparticles displaying poly(N-isopropylacrylamide) (pNIPAm), and (ii) an elastin-like polypeptide (ELP) fused with the affinity protein Cohesin (Coh). Proper design of pNIPAm-mNPs and ELP-Coh allowed for efficient cross-aggregation of the two distinct nanoparticle types under collapsing stimuli, which enabled magnetic separation of ELP-Coh aggregates bound to target Dockerin (Doc) molecules. Selective resolubilization of the ELP-Coh/Doc complexes was achieved under intermediate conditions under which only the pNIPAm-mNPs remained aggregated. We show that ELP-Coh is capable of magnetically separating and purifying nanomolar quantities of Doc as well as eukaryotic whole cells displaying the complementary Doc domain from diluted human plasma. This modular system provides magnetic enrichment and purification of captured molecular targets and eliminates the requirement of biofunctionalization of magnetic nanoparticles to achieve bioseparations. Our streamlined and simplified approach is amenable for point-of-use applications and brings the advantages of ELP-fusion proteins to the realm of magnetic particle separation systems.
Subject(s)
Bacterial Proteins/chemistry , Elastin/chemistry , Magnetite Nanoparticles/chemistry , Peptides/chemistry , Receptors, Peptide/chemistry , Recombinant Fusion Proteins/chemistry , Acrylic Resins/chemistry , Bacterial Proteins/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Separation , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Clostridium thermocellum/chemistry , Humans , Hydrogen-Ion Concentration , Kinetics , Peptides/genetics , Protein Domains , Receptors, Peptide/genetics , Recombinant Fusion Proteins/genetics , Yeasts/cytology , CohesinsABSTRACT
BACKGROUND: Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11ß-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11ß-HSD1 inhibitors. RESULTS: The yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11ß-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11ß-HSD1 inhibitory activity. As proof of concept the correlation between 11ß-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11ß-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11ß-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11ß-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times. CONCLUSIONS: Our yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11ß-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11ß-HSD1 inhibitors at intracellular level.