title: Preparation and preliminary characterization of recombinant neurolysin for in vivo studies.
abstract: The goal of this study was to produce milligram quantities of pure, catalytically active, endotoxin-free recombinant neurolysin (rNln) in standard laboratory conditions for use as a research tool. To this end, we transformed E. coli cells with a plasmid construct for polyhistidine-tagged rNln, selected a high-expressing clone and determined the optimal time-point for translation of rNln. rNln was purified to homogeneity from the soluble pool of the cell lysate using Ni-NTA affinity and size-exclusion chromatography, followed by removal of endotoxins. Using this protocol ∼3mg pure, catalytically active and nearly endotoxin-free (≈0.003EU/μg protein) rNln was reproducibly obtained from 1l of culture. Lack of cytotoxicity of rNln preparation was documented in cultured mouse cells, whereas stability in whole mouse blood. Intraperitonealy administered rNln in mice reached the systemic circulation in intact and enzymatically active form with Tmax of 1h and T1/2 of ∼30min. Administration of rNln (2 and 10mg/kg) did not alter arterial blood pressure, heart rate, body temperature and blood glucose levels in mice. These studies demonstrate that the rNln preparation is suitable for cell culture and in vivo studies and can serve as a research tool to investigate the (patho)physiological function of this peptidase.
authors: Department of Pharmaceutical Sciences, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States; College of Science and General Studies, Alfaisal University, Riyadh, 11533, Saudi Arabia.
authors: Naomi J
authors: NJ
authors: Wangler
authors: Department of Pharmaceutical Sciences, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States.
authors: Srinidhi
authors: S
authors: Jayaraman
authors: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409, United States.
authors: Rui
authors: R
authors: Zhu
authors: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409, United States.
authors: Yehia
authors: Y
authors: Mechref
authors: Department of Pharmaceutical Sciences, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States; Center for Blood Brain Barrier Research, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States.
authors: Thomas J
authors: TJ
authors: Abbruscato
authors: Department of Pharmaceutical Sciences, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States; Center for Blood Brain Barrier Research, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States.
authors: Ulrich
authors: U
authors: Bickel
authors: Department of Pharmaceutical Sciences, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States; Center for Blood Brain Barrier Research, School of Pharmacy, TTUHSC, Amarillo, TX, 79106, United States. Electronic address: vardan.karamyan@ttuhsc.edu.
authors: Vardan T
authors: VT
authors: Karamyan
mesh_terms: D000818:Animals; D001253:Astrocytes; D001921:Brain; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004731:Endotoxins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006639:Histidine / Q000737:chemistry; D008666:Metalloendopeptidases / Q000008:administration & dosage / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009474:Neurons; D055550:Protein Stability; D051381:Rats; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D004731:Endotoxins; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D008666:Metalloendopeptidases; C062316:neurolysin