Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus.
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.
Pecenková
Tamara
T
Department of Virology, Institute of Experimental Botany, Czech Academy of Sciences, Na Karlovce 1a, 160 00 Prague 6, Czech Republic.
Filigarová
Marie
M
Cerovská
Noemi
N
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005658:Fungi / Q000821:virology; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012328:RNA Viruses / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins