title: Intracellular release of recombinant green fluorescent protein (gfp(uv)) from Escherichia coli.
abstract: The recombinant green fluorescent protein (gfp(uv)) was expressed by Escherichia coli DH5-alpha cells transformed with the plasmid pGFPuv. The gfp(uv) was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (-70 degrees C for 15 h), by four freeze (-20 degrees C)/thaw cycles interlaid by sonication. The average content of released gfp(uv) (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (-70 degrees C) and the first, second, third and fourth freeze/thaw cycles, respectively. Superfusion on freezing was observed between -11 degrees C and -14 degrees C, after which it reached -20 degrees C at 0.83 degrees C/min.
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, SP, Brazil. tcvpenna@usp.br
authors.forename: Thereza Christina Vessoni
authors.initials: TC
authors.lastname: Penna
authors.forename: Eb
authors.initials: E
authors.lastname: Chiarini
authors.forename: Irene Alexeevna
authors.initials: IA
authors.lastname: Machoshvili
authors.forename: Marina
authors.initials: M
authors.lastname: Ishii
authors.forename: Adalberto
authors.initials: A
authors.lastname: Pessoa
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005615:Freezing; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins