Non-Chromatographic Protein Purification via Mini-Intein Cleavage
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A protease-free, column-free protein purification technology was developed through a combination of an engineered thio-responsive split-intein and a stimulus-responsive elastin-like polypeptide (ELP). The DnaE split intein from Nostoc punctiforme was engineered to catalyze C-terminal cleavage, rather than trans-splicing reaction under reducing condition at neutral pH. ELP is fused to the N-fragment of the split-intein and the protein of interest (POI) is fused to the C-fragment of the split-intein. Each fusion protein is expressed separately in E. Coli and after cell lysis, these proteins are mixed at pH8. After mixing, the N- and C- fragments of the split-intein associate with each other, linking the ELP to the POI. Salt is then added to the protein mixture to precipitate the ELP. The precipitant containing the ELP-intein-POI complex is then solubilized in a low-salt reducing buffer, inducing intein C-terminal cleavage reaction and separating POI from the intein and ELP. After intein reaction, the ELP-intein complex is precipitated again by the addition of high salt buffer, resulting in purified POI in the supernatant. Implementation of this technology in large scale applications can reduce the complexity, cost, and time required for protein purification.
Valdes, Najla (2012). Non-Chromatographic Protein Purification via Mini-Intein Cleavage. Honors and Undergraduate Research. Available electronically from https : / /hdl .handle .net /1969 .1 /154435.