Pečenková, Tamara; Filigarová, Marie; Čeřovská, Noemi
PROTEIN EXPRESSION AND PURIFICATION
41:
128-135,
2005
Klíčová slova:
Protein expression; Potato mop-top virus; Triple gene block
Abstrakt:
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.
Autoři z ÚEB: Noemi Čeřovská,
Tamara Pečenková
Čeština
English