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

Date Sep 5, 2013
Time 2:00 pm  
Location 116 Roger Adams Laboratory
Sponsor Chemical and Biomolecular Engineering
Event type Other
Views 6444

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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