RESUMO
In order to identify a promoter system for high-level expression of transgenes in hairy roots, we characterized the chimeric super-promoter fused to the translational enhancer from tobacco etch virus (TEV). Transgenic tobacco plants and hairy roots were generated with the super-promoter:TEV sequence and a modified green fluorescence protein (mGFP5) as a reporter gene. To exploit the utility of hairy root cultures as a secretion-based expression system, the signal peptide of patatin was fused to mGFP5 to direct its secretion into the culture medium. Levels of mGFP5 RNA were on average sixfold higher in hairy roots than leaves. Likewise, GFP protein levels per gram of fresh weight were at least tenfold higher in hairy roots than leaves. Furthermore, more than 10% of the recombinant protein produced in the hairy root culture system was found in the medium. Immunoblotting with anti-GFP antibodies showed two products of 27.1 and 29.9 kDa in all leaf and hairy root tissue extracts, whereas a single 27.1-kDa product was detected in the medium. Inducibility of the promoter was studied with mature leaves and 14-day (midlog phase) hairy roots. A twofold increase in mRNA levels was found immediately after wounding in both mature leaves and hairy roots, with a corresponding increase in mGFP5 protein after 24 h. Our studies demonstrate the utility of the super-promoter:TEV system for high-level expression of recombinant proteins in hairy root bioreactors.
Assuntos
Reatores Biológicos , Regulação da Expressão Gênica de Plantas/fisiologia , Nicotiana/metabolismo , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Primers do DNA/genética , Elementos Facilitadores Genéticos/genética , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Reação em Cadeia da Polimerase , Potyvirus/genética , Proteínas Recombinantes/metabolismo , Nicotiana/genética , Transgenes/genéticaRESUMO
In vitro studies demonstrate that porcine relaxin may possess various therapeutic effects. In this study, we explore the possibility of expressing porcine relaxin in transgenic tobacco. Tobacco was selected because it is a non-food, non-feed crop, and recombinant protein production can readily be scaled up. The cDNA of porcine preprorelaxin was under the regulation of two different constitutive promoters. DNA analysis by polymerase chain reaction verified that all transgenic plants contained the correct size of gene insert. Preliminary studies showed the presence of putative prorelaxin bands in both silver-stained SDS-PAGE and western blot. The results also indicated that tobacco-produced prorelaxin may not be properly processed to yield the mature relaxin.
Assuntos
Expressão Gênica , Nicotiana/genética , Nicotiana/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Relaxina/genética , Relaxina/metabolismo , Suínos/genética , Animais , Expressão Gênica/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Ricin B (RTB), the non-toxic lectin subunit of ricin, is a promising mucosal adjuvant and carrier for use in humans. RTB fusion proteins have been expressed in tobacco hairy root cultures, but the secreted RTB component of these proteins was vulnerable to protease degradation in the medium. Moreover, castor bean purified RTB spiked into tobacco hairy root culture media showed significant degradation after 24 h and complete loss of product after 72 h. Aqueous two-phase extraction (ATPE) was tested for fast recovery of RTB not only to partially purify the protein but also to improve its stability. Two different polyethylene glycol (PEG)/salt/water systems including PEG/potassium phosphate and PEG/sodium sulfate, were studied. RTB was shown to be favorably recovered in PEG/sodium sulfate systems. Statistical analysis indicated that the ionic strength of the system and the sodium sulfate concentration were important in optimizing the partition coefficient of RTB. A selectivity of almost three could be achieved for RTB in optimized systems, and RTB partitioned in the PEG-rich phase exhibited extended stability. Therefore, ATPE was shown to be effective in initial recovery/purification and stabilization of RTB and may hold promise for other unstable secreted proteins from hairy root culture.
