Phage display for engineering and analyzing protein interaction interfaces

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Phage display is the longest-standing platform among molecular display technologies. Recent developments have extended its utility to proteins that were previously recalcitrant to phage display. The technique has played a dominant role in forming the field of synthetic binding protein engineering, where novel interfaces have been generated from libraries built using antibody fragment frameworks and also alternative scaffolds. Combinatorial methods have also been developed for the rapid analysis of binding energetics across protein interfaces. The ability to rapidly select and analyze binding interfaces, and compatibility with high-throughput methods under diverse conditions, makes it likely that the combination of phage display and synthetic combinatorial libraries will prove to be the method of choice for synthetic binding protein engineering for broad applications.

Introduction

Most biological processes depend on molecular recognition mediated by proteins [1, 2, 3], and the ability to manipulate such interactions has proven instrumental for both basic biological research and for the development of therapeutics [4, 5]. Thus, there is great interest in the development of methods for the engineering of binding proteins with novel functions. As new molecular interaction partners are rapidly identified, there is an increasing need for methods that allows for rapid, quantitative dissection of interface energetics.

While monoclonal antibodies produced by hybridoma technology have traditionally been the predominant source of affinity reagents for biological research, the so-called ‘display’ technologies have provided powerful alternative systems. While several alternative display platforms are now available [6, 7, 8, 9, 10, 11, 12], phage display represents the earliest and still dominant system [13]. In phage display, polypeptides are displayed on the surfaces of filamentous bacteriophage particles which also contain the encoding DNA, thus establishing unambiguous phenotype-genotype linkage (Figure 1).

Recently, advances in our understanding of protein structure and function have enabled the development of libraries of ‘synthetic’ binding proteins with antigen-binding sites constructed entirely from man-made diversity. Libraries of this type have achieved complete independence from the natural immune system and hold considerable promise for further expanding the utility of in vitro selection technologies. The synthetic approach allows for the use of scaffolds that are optimized for stability and performance, including highly stable antibody frameworks and a number of promising alternative scaffolds. The synthetic approach also allows for the incorporation of modular design principles that enable facile purification, reformatting and affinity maturation. In addition, library designs can be focused for particular applications, and the results from existing libraries can be used to further improve synthetic repertoires. The ability to not only use libraries to obtain binding proteins, but also to understand binding interactions, bodes well for the future of synthetic protein libraries, since it guarantees that the technologies will continue to improve as accumulated knowledge is incorporated into new library designs.

In this review, we focus on synthetic binding protein libraries built on structurally well-characterized scaffolds with hydrophobic cores typical of globular proteins and technological advances relevant to this class of molecules. Other types of libraries, including those of random peptides or disulfide-constrained scaffolds, have also been used and have been extensively reviewed elsewhere [14, 15, 16, 17, 18]. We describe advances in the understanding of protein secretion that promise to greatly expand the diversity and quality of libraries that can be constructed in the phage display format. We also highlight synthetic antibodies and other scaffolds for which libraries have yielded not only binding proteins, but also, high resolution structures that shed light on the principles underlying molecular recognition. Finally, we describe studies that use phage-displayed libraries to provide a quantitative understanding of the energetics operating at binding interfaces. We hope that the reader will be convinced of the considerable success of such structure-based library approaches, and also, of the considerable promise of future research in these areas.

Section snippets

New phage display systems with alternative secretion pathways

In the filamentous phage display systems, proteins are fused to phage coat proteins (Figure 1) and are secreted into the periplasm. The conventional systems use signal sequences for the SecB-mediated posttranslational secretion pathway in which proteins are threaded through the membrane in an unfolded state. While this mechanism is suitable for proteins that are marginally stable in the bacterial cytoplasm, such as antibody fragments, it presents a major bottleneck for the display of proteins

Synthetic antibodies

One of the most powerful applications of phage display technology has been the transfer of natural antibody repertoires, in the form of PCR-amplified DNA from immune tissues, onto phage for in vitro selections [25, 26, 27, 28, 29]. Naïve natural repertoires of this type have been proven effective in generating antibodies against numerous antigens and have alleviated some of the major limitations of the hybridoma technology. Most importantly, the phage display system provides access to the

Engineering binding proteins using alternative scaffolds

Although synthetic and natural antibody libraries are robust technologies, alternative molecular scaffolds have also been developed. These scaffolds are mostly single-domain proteins with high stability that can tolerate extensive surface mutations for construction of a binding interface. Advances in our understanding of the factors governing molecular recognition and in selection technologies have resulted in successful development of synthetic binding proteins that recognize antigens with

Combinatorial analysis of energetics at binding interfaces

Recent studies have established the utility of phage display in quantitative energetic analysis. In this type of application, phage display is used in conjunction with highly restricted libraries to produce a statistically significant database of functional variants, as opposed to identifying a small number of variants from a library of a practically infinite size. Phage display provides a means to sort a large number of variants in an efficient and unbiased fashion so that a high-quality

Future perspectives

The success of synthetic antibodies and alternate scaffolds indicates that, with proper library design and selection methods, it is possible to produce binding proteins with virtually any framework that can present an interface of a reasonable size and shape diversity. With the diverse array of well-established scaffolds, research efforts should probably be directed to establishing significant and specific applications for each of these systems instead of developing yet another scaffold. We

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

Acknowledgements

We thank David Wood for help with figures. SK acknowledges support from the National Institute of Health (R01-GM72688 and U54 GM74946).

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