Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles.
Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.
Chisso Corporation Yokohama Research Center, 5-1 Ookawa, Kanazawa-ku, 236-8605 Yokohama, Japan.
H
H
Furukawa
R
R
Shimojyo
N
N
Ohnishi
H
H
Fukuda
A
A
Kondo
D001360:Avidin / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019921:Biotinylation; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D005427:Flocculation; D049452:Green Fluorescent Proteins; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D018189:Immunomagnetic Separation / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008863:Microspheres; D036103:Nanotechnology; D019151:Peptide Library; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013535:Suspensions; D013696:Temperature
D001426:Bacterial Proteins; C087578:FLO1 protein, S cerevisiae; D007141:Immunoglobulin Fc Fragments; D008164:Luminescent Proteins; D037241:Mannose-Binding Lectins; D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013535:Suspensions; D001360:Avidin; D049452:Green Fluorescent Proteins