Anti-EMP1 (full length) polyclonal antibody

The development of a new class of erythropoietin mimetic agents (EMA) for treating anemic conditions has been initiated with the discovery of oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus stimulating erythropoiesis. The most promising amino acid sequences have been mounted on various different polymeric structures or carrier molecules to obtain highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic profiles. Concomitant with creating new therapeutic options, erythropoietin mimetic peptide (EMP)-based drug candidates represent means to artificially enhance endurance performance and necessitate coverage by sports drug testing methods. Therefore, the aim of the present study was to develop a strategy for the comprehensive detection of EMPs in doping controls, which can be used complementary to existing protocols. Three model EMPs were used to provide proof-of-concept data. Following EPO receptor-facilitated purification of target analytes from human urine, the common presence of the cysteine-flanked core structure of EMPs was exploited to generate diagnostic peptides with the aid of a nonenzymatic cleavage procedure. Sensitive detection was accomplished by targeted-SIM/data-dependent MS2 analysis. Method characterization was conducted for the EMP-based drug peginesatide concerning specificity, linearity, precision, recovery, stability, ion suppression/enhancement, and limit of detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating the multi-analyte testing capability of the presented approach.

Erythropoietin mimetic peptide 1 (EMP1) is a 20-amino-acid cyclic peptide that can increase its erythropoietic activity approximately 10-100 times through dimerization. In the present work, an EMP1 dimer was expressed in a recombinant Escherichi coli system by fusing two EMP1 genes linked by thioredoxin (TrxA) or disulfide bond formation protein A (DsbA). Both of them were also linked with a His(6) tag to simplify the subsequent purification processes. The resulting fusion protein, two-EMP1s-in-series, was a homodimer connected by a polyglycine linker of variable length. The results showed that thioredoxin facilitated the expression and folding of two-EMP1s-in-series more properly than DsbA. The highest soluble expression levels were 0.83 g/L for TrxA-EMP1-EMP1 (S), 0.82 g/L for TrxA-EMP1-EMP1 (L) and 0.43 g/L for DsbA-EMP1-EMP1 (S). After being released from the fusion protein construct by enterokinase cleavage and subsequent reversed-phase chromatographic purification, the erythropoietic activity of two-EMP1s-in-series was 10-fold higher than that of monomeric EMP1. The length of the linker peptide between the two EMP1 peptides had a negligible effect on the erythropoietic activity. (C) 2015 Published by Elsevier Ltd.