Preparation and preliminary characterization of recombinant neurolysin for in vivo studies.

摘要: The goal of this study was to produce milligram quantities pure, catalytically active, endotoxin-free recombinant neurolysin (rNln) in standard laboratory conditions for use as a research tool. To end, we transformed E. coli cells with plasmid construct polyhistidine-tagged rNln, selected high-expressing clone and determined the optimal time-point translation rNln. rNln purified homogeneity from soluble pool cell lysate using Ni-NTA affinity size-exclusion chromatography, followed by removal endotoxins. Using protocol ∼3mg active nearly (≈0.003EU/μg protein) reproducibly obtained 1l culture. Lack cytotoxicity preparation documented cultured mouse cells, whereas stability whole blood. Intraperitonealy administered mice reached systemic circulation intact enzymatically form Tmax 1h T1/2 ∼30min. Administration (2 10mg/kg) did not alter arterial blood pressure, heart rate, body temperature glucose levels mice. These studies demonstrate that is suitable culture vivo can serve tool investigate (patho)physiological function peptidase.