Biomedical Engineering Reference
In-Depth Information
Initial gene library
Protein 2 Protein 3
Protein 1
1
2
3
Phage replication in
E. coli
and
1
2
3
1
2
3
surface expression of inserted gene
product
Phage DNA
Affinity
screening
Unmodified phages
Recombinant
production of gene
product
2
2
Recovery of
desired gene
Phage elution
Immobilized
target molecule
Figure 13.2
Phage display technology and the screening of a library for a protein capable of binding to a de-
sired target molecule. In this simplifi ed example an initial library of three genes is inserted into three phages.
One (gene 2) codes for the desired protein. In reality, libraries typically consist of hundreds of thousands/mil-
lions of different genes. Refer to text for further details
ligand with the highest specifi city/affi nity. Once this is achieved, the gene coding for the protein
of interest can be excised from the phage genome by standard techniques of molecular biology. It
can then be incorporated into an appropriate microbial/animal cell/transgenic expression system
(Chapter 3), facilitating large-scale production of the gene product. Variations of the phage display
approach have been developed, some of which utilize engineered phage (phagemids), whereas
others achieve library expression not on the surface of phage but on the surface of bacteria.
Amongst the fi rst and still most prominent application of phage display technology is the pro-
duction and screening of antibody libraries in order to isolate/identify an antibody capable of bind-
ing to a desired target molecule. As such, this technology has now come to the fore in identifying
antibodies suited to clinical application.
Genes/cDNA libraries coding for antibodies/antigen-binding antibody fragments have been ob-
tained from human and animal (e.g. mice, rabbit and chicken) sources. Two types of library can
be generated. 'Immune libraries' are obtained by cloning antibody/antibody fragment coding se-
quences derived from B-lymphocytes (usually from spleens) of donors previously immunized with
the target antigen. A high number of hits (positive clones) should be obtained from such libraries.
Non-immune libraries are produced in a similar fashion, but using B-lymphocytes from non-im-
munized donors as a source of antibody genes. This approach becomes necessary if initial immuni-
zation with the antigen of interest is not possible (e.g. due to ethical considerations). Although such
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