Čeština
EnglishEfficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus
Pečenková, Tamara; Filigarová, Marie; Čeřovská, Noemi
PROTEIN EXPRESSION AND PURIFICATION 41: 128-135, 2005
Keywords: Protein expression; Potato mop-top virus; Triple gene block
Abstract: 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 vreate a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-lenght clone by ewonuclease 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 sbe 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 (TGPp1) which was very hard toexpress in bacteria in its original length. The TGPp1 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 lysare confirmed the efficient expression of this deleted 6xHis tagged TGBp1 fragment.
DOI:
IEB authors: Noemi Čeřovská, Tamara Pečenková
PROTEIN EXPRESSION AND PURIFICATION 41: 128-135, 2005
Keywords: Protein expression; Potato mop-top virus; Triple gene block
Abstract: 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 vreate a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-lenght clone by ewonuclease 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 sbe 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 (TGPp1) which was very hard toexpress in bacteria in its original length. The TGPp1 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 lysare confirmed the efficient expression of this deleted 6xHis tagged TGBp1 fragment.
DOI:
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