Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases

Colloids and Surfaces. B, Biointerfaces
Maryam Raeeszadeh-SarmazdehEric T Boder

Abstract

We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.

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Citations

Jun 10, 2016·Journal of the American Chemical Society·Jeff E GlasgowJennifer R Cochran
Nov 11, 2017·Chembiochem : a European Journal of Chemical Biology·Keyvan D NikghalbJohn M Antos
Oct 28, 2016·F1000Research·Morten Meldal, Sanne Schoffelen
Dec 19, 2017·Journal of Biomaterials Applications·Zhi LiZhenzhong Zhang
Jun 17, 2020·Biotechnology and Bioengineering·Zhanglin LinYanrui Ye
Jul 25, 2021·The Journal of Biological Chemistry·Isabel M PiperJeanine F Amacher
Oct 3, 2019·Bioconjugate Chemistry·Zhi ZouUlrich Schwaneberg

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