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A cleavable silica‐binding affinity tag for rapid and inexpensive protein purification
Author(s) -
Coyle Brandon L.,
Baneyx François
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25257
Subject(s) - chemistry , chromatography , tandem affinity purification , maltose binding protein , affinity chromatography , linker , proteolysis , biochemistry , fusion protein , protein tag , elution , protease , fast protein liquid chromatography , protein purification , high performance liquid chromatography , recombinant dna , enzyme , computer science , gene , operating system
We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C‐terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L ‐lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9‐tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80–90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker‐Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C‐terminal extension of GFPmut2‐Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10‐fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C‐terminally‐tagged versions. Biotechnol. Bioeng. 2014;111: 2019–2026. © 2014 Wiley Periodicals, Inc.