We have combined Ligation Independent Cloning (LIC) with a self-cleaving intein to create a more efficient method for protein expression and purification.  LIC is a DNA recombination method that generates long compatible overhangs to allow the insertion of a DNA fragment into an expression vector without the need for target-specific restriction digests or ligation.  The ?I-CM self-cleaving intein creates an affinity tag-intein-target protein gene fusion, where the intein cleaves the tag from the purified target protein with a mild pH or temperature shift; thus obviating the use of expensive proteases to release the protein from the purification tag. Furthermore, the intein can be combined with a variety of purification tags, including the elastin-like polypeptide tag, for non-chromatographic purification. Through this newly developed system, arbitrary protein genes can be incorporated into various expression vectors, and their encoded proteins purified, in a non-chromatographic, high-throughput manner.

High-throughput protein purification using self-cleaving intein and ligation independent cloning

243rd National Meeting (2012)

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