The first phage displaying a protein fragment was made by inserting a foreign sequence into the otherwise wild-type genome of a filamentous phage (Smith, 1985). However, working with phage has a number of disadvantages, such as low yields of DNA and the need to work with plaques instead of colonies. A very popular alternative is to use a phagemid instead. This is a plasmid carrying a filamentous phage origin of replication that allows it to replicate and be packaged in the presence of helper phage. Generally, a helper phage is tagged with antibiotic resistance and is partially replication deficient, allowing preferential replication and packaging of the phagemid [e.g., M13K07, carrying a kanamycin resistance marker (Vieira and Messing, 1987)]. pUC119 is a common phagemid on which a large number of current display vectors, including our own, are based (Vieira and Messing, 1987). The vector we use for display, pDISPLAY-B (Fig. 3), was derived from vector pHENl (Hoogenboom et al, 1991). It carries a gene III fusion cassette, into which gene X can be inserted. As mentioned in Section II,A, proteins can be displayed as either protein III or protein VIII fusions. Phagemids carrying gene VIII fusions allow the display of many copies of the fusion protein along the full length of the viral particle, whereas phagemids carrying gene III fusions will lead to phage with very few copies (mostly one or fewer; see below) at the tip of the virion (Bass et al, 1990). When trying to select for RNA binding proteins that can bind tightly and selectively to a specific RNA target, it is best to avoid multivalent phage. Therefore, a gene Ill-type vector is used. Vectors of the gene VIII type will not be further discussed.
The fusion gene on pDISPLAY-B is controlled by the lac promoter and carries a signal sequence at its 5' end. This is followed by a polylinker region for insertion of gene X, a hexahistidine tag for protein purification, a MYC epitope tag, and the fd phage gene III. Bacteria carrying this vector produce virions only signal sequence ^ lac promoter —
His tag MYC tag
M13 origin n Sfi I Ncoi Pst\ Sal I
GTG.GCC.GCA.GAA.CAA.AAA.CTC.ATC.TCA.GAA.GAG.GAT.CTG.AAT.GGG. _gene III_
Figure 3 The phage display vector pDISPLAY-B. (A) Schematic diagram of the pDISPLAY-B phagemid. This vector was derived from phagemid pHENl (Hoogenboom et ah, 1991). Aside from a plasmid origin of replication, an ampicillin resistance gene, and the M13 origin of replication, the vector carries a gene III fusion cassette into which foreign genes can be inserted. The fusion cassette consists of the lac promoter, a signal sequence, a polylinker region, a hexahistidine tag (used for protein purification after mutants of interest have been transplanted to expression vectors), a MYC epitope tag (so that a single antibody can be used to recognize different fusion protein products), and gene III from bacteriophage fd. Restriction sites of interest are marked, with unique sites shown in boldface. (B) The polylinker and tag region of pDISPLAY-B. Unique restriction sites are shown in boldface. The signal sequence cleavage site is marked by a downward arrow. The hexahistidine tag, MYC epitope tag, and beginning of gene III are marked. The correct reading frame is indicated by dots between the codons.
after they have been infected with helper phage. The produced particles contain either a helper phage genome or a phagemid in their interior and carry a mix of wild-type protein III (from the helper phage) and fusion protein III (from the phagemid) on their exterior. Of these, only particles that can bind to RNA and yield ampicillin-resistant colonies are studied. These particles, which carry the RNA binding protein on their exterior and the phagemid in their interior, will be referred to as display phage. All other particles are counterselected, either during the binding assay or during growth on selective plates. Aside from reducing the chance of getting "multivalent" phage, the mix of wild-type and fusion protein III ensures the full infectivity of the particles, since fusions to the gene III protein have been known to affect attachment.
So far as I know, there is no limit to the size of the fragment that can be cloned into pDISPLAY-B. Possibly very large inserts might lead to unstable virions. The largest fragment we have inserted to date is a 1.4-kb fragment from the yeast poly (A)-binding protein gene (Sachs et al., 1986), encoding four RRM domains and part of the C-terminal end of the protein (also see Section V,B,4).
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