Presentation Title

Reducing the Reversibility of Sortase Mediated Ligation on Full-Sized Proteins

Presentation Type

Poster

Abstract

The Sortase A enzyme is a powerful protein engineering tool which allows for site-specific modification of molecules baring a five-amino acid recognition motif, LPXTG. Applicability is reduced, however, due to the inherent reversibility of the Sortase ligation reaction. Regeneration of the recognition motif allows for the product molecule to be targeted by Sortase, and the previously excised fragment can replace the originally intended addition. This typically leads to low product yields which can be manipulated using an excess of nucleophile, but this technique is not always practical when dealing with expensive reagents. To correct the issue, a metal assisted ligation method is being investigated. It has been previously shown that Ni2+ ions coordinate with N-terminal GGH motifs, and so the Sortase recognition sequence was expanded to LPXTGGH. When Sortase cleaves the threonine-glycine bond, the excised fragment can then coordinate with Ni2+ and is sequestered from the reaction, no longer able to participate in product reversal. This technique has been shown effective in the context of small molecule substrates and nucleophiles, as well as full sized proteins such as GFP, affibodies, and DARPINs. Efforts are also being made to implement this method to optimize the synthesis of hemoglobin tetramers.

Start Date

6-5-2017 12:15 PM

End Date

6-5-2017 2:00 PM

Location

Miller Hall

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May 6th, 12:15 PM May 6th, 2:00 PM

Reducing the Reversibility of Sortase Mediated Ligation on Full-Sized Proteins

Miller Hall

The Sortase A enzyme is a powerful protein engineering tool which allows for site-specific modification of molecules baring a five-amino acid recognition motif, LPXTG. Applicability is reduced, however, due to the inherent reversibility of the Sortase ligation reaction. Regeneration of the recognition motif allows for the product molecule to be targeted by Sortase, and the previously excised fragment can replace the originally intended addition. This typically leads to low product yields which can be manipulated using an excess of nucleophile, but this technique is not always practical when dealing with expensive reagents. To correct the issue, a metal assisted ligation method is being investigated. It has been previously shown that Ni2+ ions coordinate with N-terminal GGH motifs, and so the Sortase recognition sequence was expanded to LPXTGGH. When Sortase cleaves the threonine-glycine bond, the excised fragment can then coordinate with Ni2+ and is sequestered from the reaction, no longer able to participate in product reversal. This technique has been shown effective in the context of small molecule substrates and nucleophiles, as well as full sized proteins such as GFP, affibodies, and DARPINs. Efforts are also being made to implement this method to optimize the synthesis of hemoglobin tetramers.