title: Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation.
abstract: Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes. Thus, RNase-neutralized pancreatic membrane fractions from pig, cow, or sheep are a cheap, easily accessible, and fulfilling alternative to canine microsomes.
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven-University of Leuven, B-3000 Leuven, Belgium. Electronic address: kurt.vermeire@rega.kuleuven.be.
authors.forename: Kurt
authors.initials: K
authors.lastname: Vermeire
authors.affiliation: Institute of Biology, Center for Structural and Cell Biology in Medicine (CSCM), University of Lübeck, 23538 Lübeck, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Allan
authors.affiliation: Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, KU Leuven-University of Leuven, B-3000 Leuven, Belgium.
authors.forename: Becky
authors.initials: B
authors.lastname: Provinciael
authors.affiliation: Institute of Biology, Center for Structural and Cell Biology in Medicine (CSCM), University of Lübeck, 23538 Lübeck, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Hartmann
authors.affiliation: Institute of Biology, Center for Structural and Cell Biology in Medicine (CSCM), University of Lübeck, 23538 Lübeck, Germany.
authors.forename: Kai-Uwe
authors.initials: KU
authors.lastname: Kalies
mesh_terms: D000818:Animals; D002417:Cattle; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D010179:Pancreas / Q000166:cytology*; D021381:Protein Transport / Q000187:drug effects; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D012756:Sheep; D013552:Swine
chemical_list: D004791:Enzyme Inhibitors; D012260:Ribonucleases
keywords: Cell-free protein translation; Cyclotriazadisulfonamide (CADA); Mammalian ER membranes; Pancreatic microsomes; Ribonuclease; Translocation inhibitor