Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology

Protein Expression and Purification
Jessica Soto-RodríguezFrançois Baneyx

Abstract

Car9, a dodecapeptide identified by cell surface display for its ability to bind to the edge of carbonaceous materials, also binds to silica with high affinity. The interaction can be disrupted with l-lysine or l-arginine, enabling a broad range of technological applications. Previously, we reported that C-terminal Car9 extensions support efficient protein purification on underivatized silica. Here, we show that the Car9 tag is functional and TEV protease-excisable when fused to the N-termini of target proteins, and that it supports affinity purification under denaturing conditions, albeit with reduced yields. We further demonstrate that capture of Car9-tagged proteins is enhanced on small particle size silica gels with large pores, that the concomitant problem of nonspecific protein adsorption can be solved by lysing cells in the presence of 0.3% Tween 20, and that efficient elution is achieved at reduced l-lysine concentrations under alkaline conditions. An optimized small-scale purification kit incorporating the above features allows Car9-tagged proteins to be inexpensively recovered in minutes with better than 90% purity. The Car9 affinity purification technology should prove valuable for laboratory-scale applications requi...Continue Reading

Citations

May 25, 2020·Acta Biomaterialia·Mohamed A A Abdelhamid, Seung Pil Pack
Feb 18, 2020·Protein Expression and Purification·Meng XuFrançois Baneyx
Mar 15, 2019·Langmuir : the ACS Journal of Surfaces and Colloids·Brittney HellnerFrançois Baneyx
Jul 3, 2020·Langmuir : the ACS Journal of Surfaces and Colloids·Brittney HellnerFrançois Baneyx

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