RESUMO
The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35 kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.
Assuntos
Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , CamundongosRESUMO
The immunogenicity and functional activity of antibodies raised in mice against the cyclic disulphide peptide corresponding to the variable region 2 of PorA outer membrane protein from Neisseria meningitidis strain B385 (serosubtype P1.15), displayed on filamentous phage, were evaluated. The epitope, flanked either by cysteine or cysteine and three glycine residues, was expressed as a fusion to PVIII protein from M13. Immunisation of Balb/C mice with either phage generated antibody specific responses. Sera raised against the phage exposing the cyclic peptide through the three-glycine linker recognised the native protein better than those raised against the peptide with no linker. Only the phage displaying the cyclic peptide with linker was capable of inducing antibodies with bactericidal activity. These results indicate the possibility of using phage display for conformational peptide expression for immunisation to elicit functional antibody responses.
Assuntos
Anticorpos Antibacterianos/biossíntese , Bacteriófago M13/imunologia , Neisseria meningitidis/imunologia , Biblioteca de Peptídeos , Porinas/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Reações Antígeno-Anticorpo , Bacteriófago M13/genética , Western Blotting , Epitopos/genética , Epitopos/imunologia , Feminino , Soros Imunes/metabolismo , Esquemas de Imunização , Camundongos , Camundongos Endogâmicos BALB C , Neisseria gonorrhoeae/imunologia , Porinas/administração & dosagem , Porinas/genética , Teste Bactericida do Soro/métodosRESUMO
We have characterized the binding epitopes of two monoclonal antibodies (MAbs) reacting with human Interleukin-2 (IL-2), using a phage display peptide library. The first antibody (CB-IL2.1) recognizes the sequence LSFL, amino acid 72 to amino acid 80, numbered in the IL-2. The second antibody (CB-IL2.2) binds the sequence TTFM (amino acids 101 to 104) located at the opposite site of the four-helix bundle of IL-2. Enzyme-linked immunoadsorbent assay (ELISA) and Western blot using different IL-2 protein construct expressed in bacteria and phage display demonstrate the specificities of this antibody. The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitope are accessible from the outside in the native IL-2 molecule.
Assuntos
Anticorpos Monoclonais , Epitopos/química , Interleucina-2/química , Interleucina-2/imunologia , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Sítios de Ligação/genética , Epitopos/genética , Humanos , Hibridomas/imunologia , Interleucina-2/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Conformação ProteicaRESUMO
Previous attempts to produce active human Interleukin-2 (hIL-2) in E. coli have failed, due to its aggregation in the form of cytoplasmic inclusion bodies, and the inability of the protein to enter the periplasmic export pathway, when fused to bacterial signal sequences. We have reasoned that these limitations could be overcome by introducing changes in the signal sequence and/or in some hIL-2 residues, not critical for its biological activity; and proceeded to test this hypothesis using a phagemid vector carrying the pelB secretion signal sequence, and the filamentous phage display system. Deletion of the Pro +2 in hIL-2 led to the export of a correct size (processed) molecule to the bacterial periplasm of Su- cells by the phagemid vector. However, this was achieved under growth conditions that would not favor phage assembly in Su+ strains. Changing the hydrophobic core of the leader peptide reversed this situation and allowed phage assembly and display of a pIII/hIL-2 hybrid protein in TG1 cells. The phage-displayed hIL-2 is correctly folded, as judged by its ability to interact with a conformation-specific anti-hIL-2 monoclonal antibody, and maintains its biological activity when tested in a CTLL-2 cell proliferation assay. The changes introduced in hIL-2 and the signal sequence will make possible to use the powerful phage display technology for the selection of high-affinity variants from libraries of hIL-2 mutants.
Assuntos
Bacteriófagos/genética , Bacteriófagos/metabolismo , Interleucina-2/biossíntese , Interleucina-2/genética , Sequência de Aminoácidos , Animais , Divisão Celular/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interleucina-2/farmacologia , Camundongos , Dados de Sequência Molecular , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.