19 cDNA Library Screening
295
toriallambda libraries (H use et al. 1989 ). Binding activities expressed on the
surface of released phagemids have been identified by capturing on to immobilized ligand (biopanning) (Hogrefe et al. 1993). Protein-protein interactions can be studied in the so-called two-hybrid system (Fields and Sternglanz 1994). Screening for binding activity is not restricted to protein-protein interactions, but can also be used for identification of DNA binding
proteins ( e.g. transcription factors) as originally described by Singh et
al. (1988) and as modified since (Messier et al. 1993). cDNA libraries
may be propagated in E. coli then transferred into eukaryotic cells where
they are screened for expression. Shuttle vectors permit growth in prokaryotic as well as eukaryotic cells. In this fashion, exciting screening strategies
such as identification of expressed receptor functions have become possible
(Spengler et al. 1993).
Subcloning of desired cDNA insert - excision vectors
An additional vector system that deserves special notice is the A.ZAP expression vector (Short and Sorge 1992, Snead et al. 1997). The central distinguishing feature of this dass of vectors, called excision vectors, is the ability
to automatically excise the cDNA insert in the host cell in vivo and to convert it into so-called phagemid sequences. This in vivo excision eliminates
the need to subclone cDNA inserts and significantly reduces the time and
effort required for their analysis. Briefly, the principle is as follows: cDNA
sequences cloned into the A.ZAP bacteriophage are flanked by the initiator
and terminator sequences derived from the f1 filamentous phage origin.
Upon coinfection with M13 helper phage the M13 gene II protein is expressed which recognizes the initiator sequence of the f1 origin of replication. Subsequently single-stranded DNA is synthesized from the initiator to
the terminator including the cDNA insert, and is then circularized. The
DNA circle is packaged as fllamentous phage particles using structural proteins provided by the M13 helper phage. The packaged phagemids are extruded into the culture supernatant where they and can infect E.coli and are
propagatedas plasmids. Therefore the A.ZAP expression vector system combines the advantages of lambda phage screening with the ease of manipulating plasmid vectors.
295
toriallambda libraries (H use et al. 1989 ). Binding activities expressed on the
surface of released phagemids have been identified by capturing on to immobilized ligand (biopanning) (Hogrefe et al. 1993). Protein-protein interactions can be studied in the so-called two-hybrid system (Fields and Sternglanz 1994). Screening for binding activity is not restricted to protein-protein interactions, but can also be used for identification of DNA binding
proteins ( e.g. transcription factors) as originally described by Singh et
al. (1988) and as modified since (Messier et al. 1993). cDNA libraries
may be propagated in E. coli then transferred into eukaryotic cells where
they are screened for expression. Shuttle vectors permit growth in prokaryotic as well as eukaryotic cells. In this fashion, exciting screening strategies
such as identification of expressed receptor functions have become possible
(Spengler et al. 1993).
Subcloning of desired cDNA insert - excision vectors
An additional vector system that deserves special notice is the A.ZAP expression vector (Short and Sorge 1992, Snead et al. 1997). The central distinguishing feature of this dass of vectors, called excision vectors, is the ability
to automatically excise the cDNA insert in the host cell in vivo and to convert it into so-called phagemid sequences. This in vivo excision eliminates
the need to subclone cDNA inserts and significantly reduces the time and
effort required for their analysis. Briefly, the principle is as follows: cDNA
sequences cloned into the A.ZAP bacteriophage are flanked by the initiator
and terminator sequences derived from the f1 filamentous phage origin.
Upon coinfection with M13 helper phage the M13 gene II protein is expressed which recognizes the initiator sequence of the f1 origin of replication. Subsequently single-stranded DNA is synthesized from the initiator to
the terminator including the cDNA insert, and is then circularized. The
DNA circle is packaged as fllamentous phage particles using structural proteins provided by the M13 helper phage. The packaged phagemids are extruded into the culture supernatant where they and can infect E.coli and are
propagatedas plasmids. Therefore the A.ZAP expression vector system combines the advantages of lambda phage screening with the ease of manipulating plasmid vectors.