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 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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Pecenková T,Filigarová M,Cerovská Ndoi
10.1016/j.pep.2004.12.013keywords:
subject
Has Abstractpub_date
2005-05-01 00:00:00pages
128-35issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(04)00411-5journal_volume
41pub_type
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