In vivo selection of single-chain antibodies using a yeast two-hybrid system.
The current methodology for screening libraries of single-chain fragments of immunoglobulin variable domains (sFvs) utilizes bacterial phage systems. We have developed a unique in vivo selection protocol combining a modified yeast two-hybrid assay with a novel prey vector expressing sFvs. The viability of the system is demonstrated with the screen of a sFv library cloned into a yeast two-hybrid prey vector for molecules that target the bait ATF-2, a member of the CREB/ATF family of transcriptional regulatory proteins. The isolated sFv was capable of recognizing ATF-2 in vitro on Western blots and in vivo in mammalian cells.
Pörtner-Taliana
A
A
Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, USA. ataliana@invitrogen.com
Russell
M
M
Froning
K J
KJ
Budworth
P R
PR
Comiskey
J D
JD
Hoeffler
J P
JP
D051698:Activating Transcription Factor 2; D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000032:analysis* / Q000276:immunology; D012441:Saccharomyces cerevisiae; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology*; D020798:Two-Hybrid System Techniques
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D014157:Transcription Factors