title: Engineered mammalian vector to express EGFP-tagged proteins as biomarkers.
abstract: Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvβ3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvβ3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.
authors.affiliation: Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP 05503-900, Brazil. gsmaga@butantan.gov.br
authors.forename: Geraldo Santana
authors.initials: GS
authors.lastname: Magalhães
authors.forename: Juliana Branco
authors.initials: JB
authors.lastname: Novo
authors.forename: Patricia Bianca
authors.initials: PB
authors.lastname: Clissa
authors.forename: Maisa Splendore
authors.initials: MS
authors.lastname: Della Casa
authors.forename: Diego
authors.initials: D
authors.lastname: Butera
authors.forename: Ana Maria Moura
authors.initials: AM
authors.lastname: da Silva
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection; D014757:Viper Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015415:Biomarkers; D039302:Integrin alphaVbeta3; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C485184:enhanced green fluorescent protein; C057986:echistatin; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C099488:TEV protease