The versatility of sortase mediated ligations as a protein modification technique has been well demonstrated, but the efficiency of these reactions suffers from inherent reversibility. A number of solutions have been proposed which introduce further limitations, such as the insertion of secondary structures to create steric hindrance in the product molecule, and techniques which limit ligations to a single terminus. A preferable methodology would include the smallest possible modification site without restricting the point of ligation. One promising solution to this issue is the expansion of the LPXTG SrtA recognition sequence to LPXTGGH, which releases a GGH motif following substrate cleavage. This minor alteration has been shown to allow complexing of the GGH fragment with Ni2+ ions, thus sequestering this component from the reaction and improving yields by minimizing reaction reversibility. Here we show an expanded scope for this metal assisted sortase mediated ligation, including the Ni2+-enhanced modification of full sized proteins with a number of commonly used fluorophores and protein modifying agents.

Driving the efficiency of sortase-mediated ligation through metal-peptide complex formation

255th National Meeting (2018)

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