title: Matrix metalloproteinase 2 fused to GFP, expressed in E. coli, successfully tracked MMP-2 distribution in vivo.
abstract: Matrix Metalloproteinases (MMPs) participate in many physiological and pathological processes. One major limitation to a better understanding of the role MMPs play in these processes is the lack of well-characterized chimeric proteins and characterization of their fluorescence. The specialized literature has reported on few constructs bearing MMPs fused to the sequence of the green fluorescent protein (GFP), but none of the described constructs have been intended for expression in bacteria or for purification and use in vivo. This work has tested a recombinant reporter protein containing the MMP-2 catalytic domain fused to GFP in terms of purification efficiency, degradation of substrates in solution and in zymograms, kinetic activity, GFP fluorescence, and GFP fluorescence in whole animals after injection of the purified and lyophilized fluorescent protein. This work has also characterized rhMMP-2 (recombinant human MMP-2) and inactive clones and used them as negative controls in experiments employing catMMP-2/GFP and rhMMP-2. To our knowledge, this is the first study that has fully characterized a chimeric protein with the MMP-2 catalytic domain fused to GFP, that has efficiently purified such protein from bacteria in a single-step, and that has obtained an adequate chimeric protein for injection in animals and tracking of MMP-2 fate and activity in vivo.
authors.affiliation: Department of Morphology, Physiology and Basic Pathology, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil; Department of Biomechanics, Medicine and Rehabilitation of the Locomotor System, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil. Electronic address: alinedeazevedo@usp.br.
authors.forename: A
authors.initials: A
authors.lastname: Azevedo
authors.affiliation: Department of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil; Institutional Capacity Building Program, Coordination of Earth Science and Ecology, Museum Paraense Emilio Goeldi (MPEG), Belem, PA, Brazil.
authors.forename: A F
authors.initials: AF
authors.lastname: Prado
authors.affiliation: Department of Morphology, Physiology and Basic Pathology, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil; Department of Biomechanics, Medicine and Rehabilitation of the Locomotor System, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
authors.forename: J P M
authors.initials: JP
authors.lastname: Issa
authors.affiliation: Department of Morphology, Physiology and Basic Pathology, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil; Department of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
authors.forename: R F
authors.initials: RF
authors.lastname: Gerlach
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D058948:Cell Tracking / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000235:genetics*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020778:Matrix Metalloproteinase 2
keywords: Cardiovascular disease; Green fluorescent protein (GFP); Matrix metalloproteinase-2 (MMP-2); Protein expression and purification