RESUMEN
To obtain strong bacterial expression of proteins that seem to be hard to express in bacteria or are highly toxic for bacteria, it is possible to create a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-length clone by exonuclease III. When a construct is equipped with the 6xHis tag, a simple colony-blot procedure can be performed and a colony giving strong and efficient expression can easily be selected for high range protein expression. We utilized this procedure to produce one of potato mop-top virus (PMTV) movement proteins, namely triple gene block protein 1 (TGBp1) which was very hard to express in bacteria in its original length. The TGBp1 gene was digested with exonuclease III and nuclease S1 from its 5' terminus, leaving 6xHis tag intact. The clone that showed the strongest signal with anti-His antibodies in colony-blot procedure was found to have 44 amino acids (of total 463) deleted. The SDS-PAGE and Western blot of high range bacterial culture lysate confirmed the efficient expression of this deleted 6xHis tagged TGBp1 fragment.
Asunto(s)
Virus de Plantas/genética , Virus ARN/genética , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Hongos/virología , Expresión Génica , Genes Virales , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Solanum tuberosum/virología , Proteínas no Estructurales Virales/químicaRESUMEN
The entire nucleotide sequence for the coding regions of a Danish PMTV isolate 54-15 was determined and compared to other known and sequenced isolates of PMTV. Many nucleotide and amino acid changes were found in parts of RNA coding for the triple gene block (TGB) proteins and in the part of the RNA coding for the read-through region of the coat protein (CP). These regions for two other isolates, the mild one 54-10 and the severe one 54-19, were sequenced. Only two amino acid changes were found to correlate with the subdivision of isolates according to symptom development into mild and severe subgroups. In addition, the phylogenetic tree was obtained suggesting the closest relationship between isolates 54-15 and 54-10. Although the sequence comparisons indicate a high genetic stability of PMTV populations, a surprising change was found in the newly sequenced isolates--the replacement of the AUG start codon of the fourth gene of the TGB encoding RNA, coding for a cystein-rich protein, by the less efficient GUG start codon.