RESUMO
Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (-44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.
Assuntos
Cistatinas/genética , Helianthus/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genéticaRESUMO
In a basic research to determine the morpho-molecular interactions of plant tissues with EcoRI DNA restriction enzyme, it was demonstrated that this protein is capable of entering the sunflower and maize leaf cells using a plant tissue-abrading material and cleaving the genomic DNA at specific sites. This was inferred from the analysis of morphological patterns of EcoRI-treated leaf areas as well as using some molecular tests, including the cleavage pattern analysis of genomic DNA isolated from treated locations followed by ligation of cleaved fragments into EcoRI site of a DNA cloning vector system. The overall results indicated that the specific restriction of genomic DNA may happen following the entrance of EcoRI protein most likely into the nucleus of plant cells.
Assuntos
Compostos Inorgânicos de Carbono/metabolismo , DNA de Plantas/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Plantas/metabolismo , Silício/metabolismo , Sítios de Ligação/genética , Transporte Biológico , DNA de Plantas/genética , Eletroforese em Gel de Ágar , Helianthus/citologia , Helianthus/genética , Helianthus/metabolismo , Células Vegetais , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas/genética , Zea mays/citologia , Zea mays/genética , Zea mays/metabolismoRESUMO
A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.
