Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus

• Published in: Protein expression and purification Vol. 41; no. 1; pp. 128 - 135
• Main Authors: Pecenkova, T, Filigarova, M, Cerovska, N
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2005
• Subjects: Amino Acid Sequence; Bacteria; Cloning, Molecular; Escherichia coli - genetics; Exonuclease III deletion; Fungi - virology; Gene Expression; Genes, Viral; Molecular Sequence Data; Plant Viruses - genetics; Plasmids - genetics; Potato mop-top virus; Protein expression; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; RNA Viruses - genetics; Sequence Deletion; Sequence Homology, Amino Acid; Solanum tuberosum - virology; Triple gene block; Viral Nonstructural Proteins - biosynthesis; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Protein expression; Potato mop-top virus; Triple gene block; Exonuclease III deletion
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.12.013

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 6×His 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 6×His 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 6×His tagged TGBp1 fragment.