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PEDS Advance Access originally published online on May 10, 2008
Protein Engineering Design and Selection 2008 21(7):413-424; doi:10.1093/protein/gzn016
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Using T7 phage display to select GFP-based binders

M. Dai1,3, J. Temirov2, E. Pesavento4, C. Kiss1, N. Velappan1, P. Pavlik1, J.H. Werner1 and A.R.M. Bradbury1

1Biosciences Division 2MPA-CINT, Los Alamos National Laboratory, Los Alamos, NM, USA 33M Drug Delivery Systems, St. Paul, MN, USA 4Unite' de Biochimie, Universite' Catholique de Louvain, Louvain-la-Neuve, Belgium

1 To whom correspondence should be addressed. E-mail: amb{at}lanl.gov

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting. These results demonstrate that specific GFP-based affinity reagents, selected from T7-based libraries, can be used in applications in which only the intrinsic fluorescence is used for detection.

Keywords: affinity reagents/GFP/phage display/T7 phage

Received March 10, 2008; revised March 10, 2008; accepted March 13, 2008.


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