Gaby Renard
Universidade Federal do Rio Grande do Sul
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Featured researches published by Gaby Renard.
Insect Biochemistry and Molecular Biology | 2000
Gaby Renard; José F. Garcia; F.C. Cardoso; Marc François Richter; Judy A. Sakanari; Luiz Shozo Ozaki; Carlos Termignoni; Aoi Masuda
A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.
Microbial Cell Factories | 2008
Ana Letícia Vanz; Gaby Renard; Mario Sergio Palma; Jocelei Maria Chies; Sérgio Luiz Dalmora; Luiz Augusto Basso; Diógenes Santiago Santos
BackgroundBiopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.ResultsHere we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.ConclusionThe recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.
Insect Molecular Biology | 2002
Gaby Renard; Flávio Alves Lara; F.C. Cardoso; Flávio Costa Miguens; Marílvia Dansa-Petretski; Carlos Termignoni; Aoi Masuda
Efforts are being undertaken to control tick infestations that cause important economic losses. A cathepsin L‐like endopeptidase of Boophilus microplus was expressed in Escherichia coli; the recombinant enzyme was capable of hydrolysing gelatin, tick vitellin and bovine haemoglobin. In this paper we focus on the expression and local of synthesis of this enzyme in the tick. RT‐PCR experiments showed that this endopeptidase is transcribed in the gut of partially engorged tick females. In immunoblotting, polyclonal antibodies against the recombinant enzyme reacted with proteins of larvae older than 5 days, of fully and partially engorged female gut. In immunolocalization experiments the enzyme was localized in probable secretory cells of the gut. Based on our findings we postulate that BmCL1 may be involved in haemoglobin degradation in the B. microplus gut. This enzyme may be used as target for the control of this parasite.
International Journal of Biological Macromolecules | 2009
Raquel Cristina Schwanke; Gaby Renard; Jocelei Maria Chies; Maria M. Campos; Eraldo Luiz Batista Junior; Diógenes Santiago Santos; Luiz Augusto Basso
Human granulocyte and macrophage colony stimulating factor (hGM-CSF) is a glycoprotein that activates and enhances the differentiation and survival of neutrophils, eosinophils and macrophages, which play a key role in the innate immune response. Here we describe the construction of the hGM-CSF encoding gene, cloning, expression in Escherichia coli, purification of recombinant hGM-CSF, N-terminal amino acid sequencing, and biological activity assay using TF-1 cells. The results presented show that the combination of experimental strategies employed to obtain recombinant hGM-CSF can yield biologically active protein, and may be useful to scaling-up production of biosimilar protein.
Journal of Microbial & Biochemical Technology | 2010
Priscila Lamb Wink; Heique Marlis Bogdawa; Gaby Renard; Jocelei Maria Chies; Luiz Augusto Basso; Di genes Santiago Santos
L-Asparaginase II from Erwinia carotovora may represent an important alternative therapy in the treatment of acute childhood lymphoblastic leukaemia, despite its promising lower glutaminase activity than Escherichia coli and Erwinia chrysanthemi L-asparaginases II, currently used in treatment of this disease. Here we describe cloning, expression, purification and determination of steady-state kinetic parameters for E. carotovora L-asparaginase II: with (AspSP) and without the signal peptide (AspMP). AspMP was purified to homogeneity by a single-step protocol with 91% yield, and AspSP by a two-step protocol with 28% yield. In addition, both enzymes presented similar high specific activities: 208.1 and 237.6 U mg-1, respectively. The Km and kcat values showed that AspMP has lower glutaminase activity than AspSP. Moreover AspMP is produced by a simpler purification protocol, and at higher yield. This process can be amenable to large scale production and be of interest to researchers and biopharmaceutical companies
Journal of Microbial & Biochemical Technology | 2010
Anne Villela Dr; Gaby Renard; Mario Sergio Palma; Jocelei Maria Chies; Sérgio Luiz Dalmora; Luiz Augusto Basso; Diógenes Santiago Santos
Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF) Instituto Nacional de Ciencia e Tecnologia em Tuberculose (INCT-TB) Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS), Porto Alegre, 90619-900
Veterinary Parasitology | 2005
Itabajara da Silva Vaz; Saiki Imamura; Chie Nakajima; F.C. Cardoso; Carlos A. Ferreira; Gaby Renard; Aoi Masuda; Kazuhiko Ohashi; Misao Onuma
Biochemical and Biophysical Research Communications | 2003
Walter Filgueira de Azevedo; Giovanni César Santos; Denis Marangoni dos Santos; Johnny Rizzieri Olivieri; Fernanda Canduri; Rafael G. Silva; Luiz Augusto Basso; Gaby Renard; Isabel Osório da Fonseca; Maria Anita Mendes; Mario Sergio Palma; Diógenes Santiago Santos
BMC Biotechnology | 2014
Laura S Marder; Juleane Lunardi; Gaby Renard; Diana C. Rostirolla; Guilherme Oliveira Petersen; José Eduardo Sacconi Nunes; Ana Paula Duarte de Souza; Ana Christina de Oliveira Dias; Jocelei Maria Chies; Luiz Augusto Basso; Diógenes Santiago Santos; Cristiano V. Bizarro
Veterinary Parasitology | 2005
I Dasilvavazjr; Sousuke Imamura; Chie Nakajima; F. M. Cardoso; Carlos A. Ferreira; Gaby Renard; Aoi Masuda; Kyoichi Ohashi; Manabu Onuma