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linked to a major coat protein of a coliphage whilst the single-stranded gene encoding

the protein is packaged within the virion. The initial steps of the method rely on the PCR

to amplify gene fragments that represent functional domains or subunits of a protein

such as an antibody. These are then cloned into a phage display vector which is an

adapted phagemid vector (Section 6.3.3) and used to transformE. coli. A helper phage is

then added to provide accessory proteins for new phage molecules to be constructed.

The DNA fragments representing the protein or polypeptide of interest are also tran-

scribed and translated, but linked to the major coat protein g III. Thus when the phage is

assembled the protein or polypeptide of interest is incorporated into the coat of the

phage and displayed, whilst the corresponding DNA is encapsulated (Fig. 6.37).

There are numerous applications for the display of proteins on the surface of bacterio-

phage viruses, bacteria and other organisms, and commercial organisations have been

quick to exploit this technology. One major application is the analysis and production of

engineered antibodies from which the technology was mainly developed. In general

phage-based systems have a number of novel applications in terms of ease of selection

rather than screening of antibody fragments, allowing analysis by methods such as

affinity chromatography. In this way it is possible to generate large numbers of antibody

heavy and light chain genes by PCR amplification and mix them in a random fashion.

Thisrecombinatorial libraryapproach may allow new or novel partners to be formed

Amplify DNA sequence by PCR

Clone fragment into phage display vector

Coat protein III

PCR

fragment

Coat protein

VIII

f1 origin

Phage surface

display vector

TransformE.coli with construct

SuperinfectE.coli with helper phage

Expression of phage gene III–insert

produces a fusion, coat protein III–protein

During phage assembly protein is displayed

on the surface whilst its DNA is phage encoded

Fig. 6.37Flow diagram indicating the main steps in the phage display technique.

239 6.7 Expression of foreign genes