Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein...

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Bibliographic Details
Published in:Nucleic acids research 1998-11, Vol.26 (22), p.5109-5115
Main Authors: Chong, Shaorong, Montello, Geoffrey E., Zhang, Aihua, Cantor, Eric J., Liao, Wei, Xu, Ming-Qun, Benner, Jack
Format: Article
Language:English
Subjects:
Affinity Labels
Base Sequence
Chromatography, Affinity - methods
Cloning, Molecular
DNA Primers - genetics
Escherichia coli - genetics
Gene Expression
Genetic Vectors
Plasmids - genetics
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
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Summary:A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/26.22.5109