Isolation of erythropoietin receptor agonist peptides using evolved phage libraries.

Cyclic peptides capable of activating the erythropoietin receptor (EPOR) were isolated from phage display libraries by screening with a novel EPOR-IgG fusion protein reagent. A parental clone ERB1 (EPO Receptor Binder 1) was first isolated from a phage display library displaying 38 random amino acids as an N-terminal fusion with the M13 minor capsid protein, pill. An evolved library was then produced from the parental sequence using an oligonucleotide saturation mutagenesis strategy which yielded EPOR binding sequences with 20 times the relative affinity of ERB1.

Expression and ligand binding assays of soluble cytokine receptor-immunoglobulin fusion proteins.

We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection.

Persistence of T-cell clones in psoriatic lesions.

Background: We previously demonstrated a clonal dominance in the V beta 13.1 messages isolated from the lesional CD8+ T cells of psoriasis vulgaris, which suggested an interaction of V beta 13.1+ CD8+ T cells with skin antigens.

Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer.

The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation.

Homogeneous detection of nucleic acids by transient-state polarized fluorescence.

We describe a transient-state polarized fluorescence-based method for detecting nucleic acids. An active ester of the phthalocyanine dye La Jolla Blue was coupled to an oligonucleotide containing an amino group at its 5' end, and the conjugate was purified by HPLC chromatography. We monitored the hybridization characteristics of the conjugate with complementary oligonucleotides and RNA as targets by transient-state polarized fluorescence measurements.