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CHBE 565 Seminar, Professor Zhilei Chen, Texas A&M University, "Engineering Inteins for Protein Hydrogel Synthesis and Protein Purification"

Event Type
Chemical and Biomolecular Engineering
116 Roger Adams Laboratory
Sep 5, 2013   2:00 pm  
Originating Calendar
Chemical & Biomolecular Engineering Seminars and Events

Inteins are protein splicing elements that can excise themselves from precursor proteins and ligate the surrounding sequences (exteins). We exploited the ultra-fast trans-splicing reaction of the naturally split DNaE intein from Nostoc punctiform  for the development of two technologies: (1) a two-component self-assembling functional protein hydrogel with potential applications in biofuel cells and tissue engineering, and (2) a generally applicable ultra-rapid tag removal step for the affinity purification of proteins. Our intein-mediated protein hydrogel utilizes two soluble protein block copolymers, each containing a subunit of a trimeric protein that serves as a crosslinker and one half of the Npu split intein.  Mixing of these two protein block copolymers initiates an intein trans-splicing reaction that reconstitutes a self-assembling polypeptide flanked by crosslinkers, triggering protein hydrogel formation. Incorporation of an appropriate binding motif into the protein block copolymers enables the convenient site-specific incorporation of functional globular proteins into the hydrogel network. Application of the Npu split intein in affinity tag removal was enabled by conversion of the intein’s trans-splicing activity into an inducible C-terminus-cleavage activity via rational protein design. We used the engineered C-terminus-cleaving split intein as a tag removal tool to purify tagless affinity-captured protein from E. coli lysate within 1 hour – the hitherto fastest reported intein technology for the purification of tagless recombinant protein.

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