ReviewPhage display in pharmaceutical biotechnology
Introduction
Phage display is a powerful technology for selecting and engineering polypeptides with novel functions. If DNA fragments encoding polypeptides are fused to certain bacteriophage coat protein genes, the fusion genes can be encapsulated within phage particles that also display the encoded polypeptides on their surfaces. This establishes a physical linkage between phenotype and genotype. Highly diverse libraries can be constructed by fusing degenerate DNA to a coat protein gene, and library members with desired binding specificities can be isolated by binding to an immobilized receptor in vitro. The sequences of selected polypeptides can be determined from the sequence of the encapsulated, encoding DNA.
Phage display was first developed with the Escherichia coli-specific bacteriophage M13 [1], and the success of M13 phage display has prompted the development of numerous alternative display systems. These include systems that utilize other E. coli-specific phage, such as λ-phage [2] and T4 phage [3], and also systems that use eukaryotic viruses [4]. In addition, polypeptides have been displayed on the surfaces of bacteria and yeast [5]. Although these alternative systems have proven advantageous in special applications, M13 phage display remains the dominant technology.
This review covers developments in M13 phage display made over the past year. I discuss technological improvements that enable the construction of larger libraries and new display formats that extend the technology to new applications. I also highlight some important applications of peptide and protein phage display, with particular emphasis on pharmaceutical biotechnology.
Section snippets
Library construction
The success of any selection experiment ultimately depends on the diversity and quality of the initial library. Over the years, methods have been refined to the point where extremely large libraries can now be rapidly and reliably constructed. Sidhu et al. [6, [7] have described optimized methods that enable the construction of libraries with diversities greater than 1012, almost 100-fold greater than previously thought practical.
Vectors and display formats
The M13 phage particle consists of a single-stranded DNA core
Applications for phage-displayed peptide libraries
Phage-displayed peptide libraries can be used to isolate peptides that bind with high specificity and affinity to virtually any target protein. These binding peptides can be used as reagents to understand molecular recognition, as minimized mimics for receptors, or as lead molecules in drug design.
Applications for phage-displayed proteins
The ability to display large proteins on M13 phage has led to numerous applications. I focus on areas that have advanced significantly in the past year and are likely to have a major impact on biotechnology.
Conclusions
Intensive efforts from many researchers have made phage display an invaluable component of biotechnology. Improved library construction methods — in combination with numerous vectors and display formats — will extend the technology even further. Phage antibodies are likely to play an even greater role in the generation of analytical reagents and therapeutic drugs. Highly diverse peptide libraries can be used to isolate specific ligands for virtually any target of interest, and these ligands
Acknowledgements
I thank Nicholas Skelton and David Wood for help with figures.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
of special interest
of outstanding interest
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