Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10843950 | Protein Expression and Purification | 2005 | 8 Pages |
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 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 6ÃHis 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 5â² terminus, leaving 6ÃHis 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 6ÃHis tagged TGBp1 fragment.
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Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Tamara PeÄenková, Marie Filigarová, Noemi ÄeÅovská,