Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture

被引:56
作者
Yeung, YA
Wittrup, KD [1 ]
机构
[1] MIT, Dept Chem Engn, Cambridge, MA 02134 USA
[2] MIT, Div Bioengn & Environm Hlth, Cambridge, MA 02134 USA
关键词
D O I
10.1021/bp010186l
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.
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收藏
页码:212 / 220
页数:9
相关论文
共 33 条
[1]   Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC) [J].
Ashcroft, RG ;
Lopez, PA .
JOURNAL OF IMMUNOLOGICAL METHODS, 2000, 243 (1-2) :13-24
[2]  
BELL GI, 1978, SCIENCE, V200, P618, DOI 10.1126/science.347575
[3]   ESTIMATE OF THE STICKING PROBABILITY FOR CELLS IN UNIFORM SHEAR-FLOW WITH ADHESION CAUSED BY SPECIFIC BONDS [J].
BELL, GI .
CELL BIOPHYSICS, 1981, 3 (03) :289-304
[4]   Optimal screening of surface-displayed polypeptide libraries [J].
Boder, ET ;
Wittrup, KD .
BIOTECHNOLOGY PROGRESS, 1998, 14 (01) :55-62
[5]   Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity [J].
Boder, ET ;
Midelfort, KS ;
Wittrup, KD .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (20) :10701-10705
[6]   Yeast surface display for screening combinatorial polypeptide libraries [J].
Boder, ET ;
Wittrup, KD .
NATURE BIOTECHNOLOGY, 1997, 15 (06) :553-557
[7]  
CAPO C, 1982, J CELL SCI, V56, P21
[8]  
Chalmers JJ, 1998, BIOTECHNOL BIOENG, V59, P10, DOI 10.1002/(SICI)1097-0290(19980705)59:1<10::AID-BIT3>3.3.CO
[9]  
2-S
[10]   The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides [J].
Christmann, A ;
Walter, K ;
Wentzel, A ;
Krätzner, R ;
Kolmar, H .
PROTEIN ENGINEERING, 1999, 12 (09) :797-806