Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology
| Autor: | Brandon L. Coyle, Ariana Samuelson, Kannan Aravagiri, Jessica Soto-Rodríguez, François Baneyx |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Strep-tag Lysis Recombinant Fusion Proteins Gene Expression Polysorbates 02 engineering and technology Arginine Chromatography Affinity beta-Lactamases 03 medical and health sciences Affinity chromatography Protein purification Endopeptidases Escherichia coli Small particles Amino Acid Sequence Cloning Molecular Tandem affinity purification Chromatography Staining and Labeling Chemistry Elution Lysine 021001 nanoscience & nanotechnology Silicon Dioxide 030104 developmental biology Proteolysis Adsorption 0210 nano-technology Oligopeptides Biotechnology Protein adsorption Plasmids |
| Zdroj: | Protein expression and purification. 135 |
| ISSN: | 1096-0279 |
| Popis: | 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 requiring rapid access to milligram-quantities of proteins, and for preparative scale purification schemes where cost and productivity are important factors. |
| Databáze: | OpenAIRE |
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