ABSTRACT
An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.
Subject(s)
Genetic Engineering/methods , Granulocyte Colony-Stimulating Factor/metabolism , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Nicotiana/genetics , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Gene Expression , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/isolation & purification , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/isolation & purification , Humans , Mice , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Nicotiana/metabolismABSTRACT
A new potato virus X (PVX)-based viral vector for superproduction of target proteins in plants has been constructed. The triple gene block and coat protein gene of PVX were substituted by green fluorescent protein. This reduced viral vector was delivered into plant cells by agroinjection (injection of Agrobacterium tumefaciens cells, carrying viral vector cDNA within T-DNA, into plant leaves), and this approach allowed to dramatically reduce the size of the vector genome. The novel vector can be used for production of different proteins including pharmaceuticals in plants.
Subject(s)
Genetic Vectors , Nicotiana/genetics , Potexvirus/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Agrobacterium tumefaciens/genetics , Base Sequence , DNA, Complementary/genetics , Genome, Viral , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Plants, Genetically Modified , Replicon , Nicotiana/metabolism , TransfectionSubject(s)
Gene Expression Regulation, Viral , Nicotiana/virology , Tobacco Mosaic Virus/growth & development , Viral Proteins/physiology , Virus Replication/genetics , Gene Silencing , Genetic Vectors/genetics , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Plant Viral Movement Proteins , Rhizobium/genetics , Nicotiana/chemistry , Tobacco Mosaic Virus/genetics , Transfection , Viral Proteins/analysis , Viral Proteins/geneticsABSTRACT
Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.
