274187 Ligation-Independent Cloning with Self-Cleaving Intein As a Tool for High-Throughput Protein Purification

Thursday, November 1, 2012: 12:30 PM
Allegheny I (Westin )
Tiana D. Warren, Michael J. Coolbaugh and David W. Wood, Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH

Ligation Independent Cloning (LIC) is being incorporated into the ΔI-CM self-cleaving intein system to create a more streamlined method for the cloning, expression and purification of proteins. In comparison to conventional molecular cloning, LIC relies on extended segments of complementary DNA for facilitating highly efficient in vivo ligation, thus eliminating the need for in vitro restriction digestion and ligation. The self-cleaving intein used for this project, ΔI-CM, was designed by the Wood lab as an alternate method for protein purification. The intein is a 168 amino acid sequence that is expressed along with the target protein and an purification tag to create a tag-intein-target protein fusion. With a mild pH or temperature shift, the intein will self-cleave from the target protein, resulting in a pure protein product, thus obviating the use of expensive proteases to release the protein from the purification tag. Through this newly developed system, different protein genes can be incorporated into various expression vectors and their encoded proteins purified in a high-throughput manner. Two vectors were constructed with the Chiting Binding Domain (CBD) and Elastin-like polypeptide (ELP) purification tags to be used for LIC and intein-mediated protein purification.  With these vectors, Maltose Binding Protein and β-lactamase proteins were successfully cloned, expressed and purified using both affinity chromatography and non-chromatographic purification.

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