The trophectoderm layer of the blastocyst-stage embryo is precursor for all trophoblast cells in placenta. Human stem (TS) have emerged as an attractive tool studies on early development. However, use TS cell models constrained by limited genetic diversity existing lines and restrictions using human fetal tissue or embryos needed to generate additional lines. Here we report derivation two distinct types lineage from pluripotent cells. Analogous villous cytotrophoblasts vivo, first a CDX2-...

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance proteolysis, and higher specificity relative linear peptides. Here we describe the discovery, synthesis characterization of novel cyclic peptide that bind Fc portion human Immunoglobulin G (IgG; hFc). We generated an mRNA display library pentapeptides wherein cyclization was achieved with high yield selectivity, using a solid-phase crosslinking reaction between two...

We present a simple method for attaching silver nanoparticles to polypropylene (PP) fibers in two-step process impart antibacterial properties. Specifically, PP are pretreated by the adsorption from an aqueous solution of heat-denatured lysozyme (LYS) followed LYS cross-linking using glutaraldehyde and sodium borohydride. At neutral pH, surface adsorbed layer is enriched with numerous positive charges. Silver (AgNPs) capped trisodium citrate subsequently deposited onto protein-coated PP....

Numerous experimental strategies exist for relative protein quantification, one of the primary objectives mass spectrometry based proteomics analysis. These mostly involve incorporation a stable isotope label via either metabolic in cell or tissue culture (15N/14N labeling, labeling by amino acids (SILAC)), chemical derivatization (ICAT, iTRAQ, TMT), enzymatically catalyzed (18O labeling). Also, these techniques can be cost time prohibitive not amenable to biological system interest (i.e.,...

Human embryonic stem cells (hESCs) are self-renewing pluripotent with relevance to treatment of numerous medical conditions. However, a global understanding the role hESC proteome in maintaining pluripotency or triggering differentiation is still largely lacking. The emergence top-down proteomics has facilitated identification and characterization intact protein forms that not readily apparent bottom-up studies. Combined metabolic labeling techniques such as stable isotope by amino acids...

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis Sso7d protein from hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated a combinatorial library mutants yeast surface display. Subsequently, we identified mutant, Sso7d-his-hFc, systematic evaluation containing single...

We have used directed evolution to construct IL-2 mutants that bind the α receptor subunit (IL-2Rα, CD25) with affinities comparable of IL-15−IL-15 (IL-15Rα) interaction. T cells proliferate for up 6 days following a 30 minute incubation these mutants, which may lead potential applications cancer and viral immunotherapy. Several alternative mechanisms been proposed explain contrasting effects IL-15 on cell proliferation death. These exhibit growth response−receptor occupancy curves...

Stimulation of T‐cells by IL‐2 has been exploited for treatment metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited negligible stimulation at low picomolar concentrations undesirable NK cells nanomolar hampers IL‐2‐based therapies. We hypothesized that increasing the affinity IL‐2Rα may create class mutants with increased biological potency as compared wild‐type IL‐2. Towards this end, we have screened libraries mutated displayed on surface yeast isolated...

We have engineered human epidermal growth factor (EGF) by directed evolution through yeast surface display for significantly enhanced affinity the EGF receptor (EGFR). Statistical analysis of improved mutants isolated from randomly mutated yeast-displayed libraries indicates that mutations are biased towards substitutions at positions exhibiting significant phylogenetic variation. In particular, in high-affinity statistically residues found orthologous species. This same trend was also...

We investigate the immobilization of a model system functionalized yeast that surface-display enhanced green fluorescent protein (eGFP) within chemically crosslinked polyvinyl alcohol (PVA) nanofibers. Yeast is incorporated into water insoluble nanofibrous materials by direct electrospinning with PVA followed vapor phase chemical crosslinking polymer. Incorporation fibers confirmed elemental analysis and viability indicated live/dead staining. Following crosslinking, we confirm maintains its...

We present the construction and screening of yeast display libraries post-translationally modified peptides wherein site-selective enzymatic treatment linear is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) followed by with recombinant transglutaminase in solution; or (ii) intracellular co-expression to achieve peptide modification endoplasmic reticulum prior surface display. The efficiency was evaluated via orthogonal detection...

Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of self-renewal differentiation. Current MS-based approaches for identifying proteins rely predominantly on MS analysis cell supernatants. Here we show that targeted proteomics secretory pathway organelles is a powerful alternate approach interrogating the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse fibroblasts...

Protein quantification is one of the principal goals mass spectrometry (MS)‐based proteomics, and many strategies exist to achieve it. Several approaches involve incorporation a stable‐isotope label using either chemical derivatization, enzymatically catalyzed 18 O, or metabolic labeling in cell tissue culture. These techniques can be cost time prohibitive not amenable biological system interest. Label‐free including those utilizing integrated ion abundance spectral counting offer an...

Microtubule cytoskeleton exists in various biochemical forms different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known affect microtubule stability, dynamics, and interaction with MAPs motors a specific manner, widely as code hypothesis. At present, there no tool that can specifically mark living cells, thus severely limiting our understanding of their dynamics cellular functions. Using yeast display library, we identified binder against terminal tyrosine...

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well characterization proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables discovery specific but also efficient, estimation their affinities (KD). In qYY2H, bait and prey are expressed yeast cell surface fusions using display. developed semiempirical framework...

The need for recombinant expression of soluble protein slows the validation engineered proteins isolated from combinatorial libraries and limits number variants evaluated. To overcome this bottleneck, we describe a system simultaneous cell surface display secretion in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two single open reading frame. Incorporation F2A peptide sequence—with ∼50% efficiency...