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Production and evaluation of parathyroid hormone receptor ligands with intrinsic or assembled peroxidase domains. | LitMetric

AI Article Synopsis

Article Abstract

Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH receptor (PTHR). We developed fusion protein ligands with enzymatic activity to probe PTHRs at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2). Alternatively, myc-tagged PTH (PTH-myc) was combined with antibodies, some of which HRP-conjugated, in the extracellular fluid. The three PTH-fusion proteins were produced as conditioned mediums (CM) by transfected producer HEK 293a cells. Binding of receptor-bound enzymatic ligands was revealed using widely available substrate/co-substrate systems. The stimulation of recipient HEK 293a expressing PTHRs with the PTH-myc/antibodies combination or with PTH-APEX2 supported the histochemical or luminescent detection of recombinant PTHRs (TrueBlue or luminol-based reagent). The PTH-HRP construction was the most sensitive and supported all tested peroxidase co-substrates (TrueBlue, tetramethylbenzidine (TMB), luminol, biotin-phenol with streptavidin-Qdots); the 3 latter schemes identified endogenous PTHR in the osteoblastic HOS cell line. The specificity of the fusion protein binding to PTHR was determined by its competition with an excess of PTH. Bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638942PMC
http://dx.doi.org/10.1038/s41598-017-13548-0DOI Listing

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