PMID:9801307
| Citation |
Chong, S, Montello, GE, Zhang, A, Cantor, EJ, Liao, W, Xu, MQ and Benner, J (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 26:5109-15 |
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| Abstract |
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. |
| Links | |
| Keywords |
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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