Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform.
Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.
Miao
Jun
J
Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Wu
Wei
W
Spielmann
Thomas
T
Belfort
Marlene
M
Derbyshire
Victoria
V
Belfort
Georges
G
D002846:Chromatography, Affinity / Q000295:instrumentation; D004706:Endodeoxyribonucleases / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D047668:Inteins / Q000502:physiology; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D019154:Protein Splicing / Q000502:physiology; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease