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As these results are described in detail in Chapter 6 of this volume, they will not be discussed here. Two early E. coli surface display formats tether the protein of interest as N-terminal fusions, such that the C-terminus is free. One of these, the Lpp'OmpA system developed by Georgiou and coworkers, is a three-part fusion protein anchored into the E. coli outer membrane (Figure 2.2A) (Francisco et al. 1992). The first component, from E. coli lipoprotein (Lpp), is a nine-amino-acid signal...

Overcoming Limitations Circumventing The Fixed Backbone

The fixed backbone simplifies the size of the design problem by reducing it to a choice of residue types and orientations along a rigid frame. It also removes the reliance on the design force field to discriminate between competing backbone structures. Since core designs start with a wild-type protein template, the choice of backbone position is obvious and likely to be reasonable, as nature has already evolved toward it. This assumption breaks down, however, when the design template is not a...

Contents

Flow Intracellular Cell Surface Compartmentalization of Whole In Vitro In Vivo Auxotroph Chemical Colony In Vitro Phage Ribosome High-Throughput Screening in Microtiter While our understanding of biological systems may one day be sufficiently comprehensive to engineer proteins in a direct and targeted fashion with predictable outcomes, biological complexity continues to stifle even seemingly simple rational engineering plans, often leaving combinatorial approaches as the most likely means of...

References

Xu, Y. Zhang and N. Tsurushita. 2007. Whole IgG surface display on mammalian cells Application to isolation of neutralizing chicken monoclonal anti-IL-12 antibodies. J Immunol Methods 327 40-52. Antipov, E., A. E. Cho, K. D. Wittrup and A. M. Klibanov. 2008. Highly L and D enantiose-lective variants of horseradish peroxidase discovered by an ultrahigh-throughput selection method. Proc Natl Acad Sci U S A 105 17694-9. Armstrong, K. A. and B. Tidor. 2008....

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FIGuRE 2.3 Representative yeast surface display platforms that have been used in protein engineering applications. The presence, position, and identity of epitope tags are variable. An example of binding interactions with fluorescently labeled target and fluorescently labeled antibodies is shown in A. Wittrup 1997 Gai and Wittrup 2007). The protein to be engineered is fused to the yeast agglutinin protein Aga2, whose expression is controlled by a galactose-inducible promoter. Aga2 is...

Engineering substrate binding properties

A number of experimental methods, such as phage display, are available for detecting and improving protein affinity for a substrate. The immune system is an example of optimized binding with antibodies that boast picomolar affinities for their cognate ligands. However, laboratory evolution and affinity maturation generally require detectable initial binding activity. A complementary role for computational design would involve constructing the binding sites de novo, which could then be enhanced...

Future Perspectives

Phage display is the longest-standing platform for the selection of protein structure and function from combinatorial libraries. The robustness of the method has made phage display a powerful tool for protein engineering, and we anticipate that automation and robotics will exponentially expand the power of the technology and enable high-throughput protein engineering. The developing fields of functional genomics and systems biology are concerned with the analysis of complete processes rather...

Successful scaffolds

Over the years, attempts have been made to use virtually all classes of protein architecture as scaffolds for constructing synthetic interfaces. While loops are the most common location into which amino-acid diversity is introduced, surface-exposed residues on a-helices or p-strands have also successfully been used. The examples presented in this section demonstrate that a synthetic interface can be engineered as long as a contiguous surface (patch) of a substantial size (> 15 residues) is...

Approaches For Computational Protein Design

Numerous search algorithms have been developed to search the energy landscape for low energy sequences and their preferred amino acids at each position. These algorithms are divided into two classes stochastic and deterministic. Stochastic algorithms use probabilistic trajectories, where the resulting sequence depends on initial conditions and a random number generator. Stochastic algorithms do not guarantee finding the GMEC sequence, but they can always find an approximate solution or a set of...

Examples Of Protein Therapeutics

The previously described protein engineering and design strategies have been applied to engineer a wide variety of protein therapeutics for enhanced activity, stability, affinity, specificity, pharmacokinetics, pharmacodynamics, reduced immunogenic-ity, and improved productivity. Extensive review articles and book volumes have been devoted to the subject of monoclonal antibodies and antibody engineering therefore, a detailed discussion will not be provided here. However, it is worth mentioning...

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FIGuRE 4.1 Major classes of library-construction methods used in protein engineering. The black and gray lines represent polypeptide sequences of the starting clone or clones, and the bullets represent mutations. (A) Random mutagenesis. (B) Recombination. (C) Site-directed diversification. (D) Scanning mutagenesis. Scanning mutagenesis (Figure 4.1D), formally a type of site-directed diversification, can be used either to generate improved protein variants or to collect sequence-function...

New classes of therapeutic proteins under development

T cells play a central role in cell-mediated immunity however, the molecular mechanism underlying TCR recognition was not well understood until the late 1980s. Unlike antibodies that can recognize pathogens or their toxins directly, T cells recognize only short peptides derived from pathogens in complex with MHC molecules on the surface of antigen presenting cells (APCs) through their TCRs. The nature of the interaction between TCRs and peptide-MHC (pMHC) complexes determines the function of...

Info

Sum(Best Energies for nj) (-2 + -2 + 1 + -3) -6 Sum(Worst Energies for nk) (-1 + -1 + 0 + -5) -7 Sum(Best Energies for nj) (-2 + -2 + 1 + -3) -6 Sum(Worst Energies for nk) (-1 + -1 + 0 + -5) -7 FIGURE 17.3 Example of dead-end elimination (DEE). The basic principle of the DEE algorithm is that some rotamers can be proven never to be in the GMEC, by identification of another rotamer to which it would always be favorable to switch. In the simplest form, rotamer j at position n (nj) can be...

Ylv

(Roberts and Szostak 1997) B. mRNA display FIGuRE 3.2 Ribosome display (A) and mRNA display (B). A simple affinity selection is pictured. (A) In ribosome display, a library of DNA constructs is transcribed in vitro. The unmodified mRNA, which contains no stop codon, is translated to form the selection particle, a stalled ribosomal complex that consists of mRNA, ribosome, and protein. These ternary complexes are allowed to bind to immobilized target molecules. Unbound or nonspecifi-cally bound...

Evolution Of Binding Agents

In the past decade, several different protein frameworks have been harnessed to derive novel binding agents for use in biotechnology and drug discovery (Figure 1.4). Antibodies represent by far the most abundant framework under development. Within antibodies, six hypervariable loops or complementarity determining regions (CDRs) define the combined site responsible for antigen recognition (Knappik et al. 2000 FIGURE 1.4 Structures of scaffolds used for developing binding agents. Positions in the...

Catalysis

Perhaps the most rigorous test of computational protein engineering is the design of functional enzymes. Catalysis by enzymes is a complex process it requires binding substrate(s), orienting the appropriate chemically reactive groups in the correct geometry, stabilizing the transition state to facilitate the chemical reaction, and releasing the product. As engineering a novel enzyme function is a very ambitious goal, it makes sense that initial attempts to design catalysts focused on the...

Vikas Nanda Fei Xu and Daniel Hsieh Contents

Designing Thermostable Stabilizing the Native Destabilizing Unfolded and Misfolded Engineering Substrate Binding Designing Proteins are malleable molecules and tolerate many mutations with marginal effects on structure and function. A limited number of three-dimensional folds are recycled repeatedly for different functions. This malleability results from surviving hundreds of millions of years of mutation and recombination. Natural proteins thus provide a gold mine of starting materials for...

Protein therapeutics versus small molecule drugs

Small molecule drugs have several advantages, including oral bioavailability, ability to reach intracellular targets, ease of manufacturing, and generally a long shelf life. These characteristics make them favorable over protein drugs in the pharmaceutical industry (see the section titled Challenges in Pharmaceutical Translation of New Therapeutic Proteins, in this chapter). However, small molecule drugs, which typically have a molecular weight less than 1000 Daltons, have limited surface area...

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Phage And Yeast Display

Phages displaying TCRs with low affinity are washed off Yeast cells with high fluorescence signal in region 1 R1 are collected Yeast cells with high fluorescence signal in region 1 R1 are collected FIGURE 7.3 see color insert following page 178 TCR engineering using A phage display and B yeast display, adapted from Boder and Wittrup 1997 . A phage display library of TCRs is usually screened by panning phage particles on a surface matrix coated with purified pMHC complexes, while a yeast display...

Geometrybased Design

There are two significant limitations to all computational design efforts 1 the extent to which the design criteria theory match reality and 2 the speed with which one can make and test proteins. In the 1990s, when computational protein design was in its infancy, fitness functions to accurately describe molecular interactions and algorithms to search the astronomical number of potential designed proteins were too primitive for the design of complex stereochemical surfaces. Furthermore, the...

Preface

Proteins possess a broad range of structural and functional properties that are unmatched by any other class of biological molecules. Amazingly, nature has arranged simple atoms and chemical bonds in such a way to facilitate complex biological processes like molecular recognition and catalysis. Nature has also inspired many scientists and engineers to design and create their own customized proteins. These engineered proteins can serve as novel molecular tools for scientific, medical, and...

Yeast Surface Display

Yeast surface display physically couples the protein to be engineered to an anchor protein embedded within the yeast cell wall. The eukaryotic secretory pathway of yeast allows a wide variety of proteins to be displayed, including many with complicated folds. Proteins that have been engineered using yeast surface display include scFvs Feldhaus and Siegel 2004 Chao et al. 2006 , Fab antibody fragments Weaver-Feldhaus et al. 2004 , single-chain T-cell receptors scTCRs Shusta et al. 1999 ,...

Faster

The fast and accurate side-chain topology and energy refinement FASTER method Desmet et al. 2002 is a heuristic iterative optimization method inspired by the previous success of the dead-end elimination DEE techniques. It attempts to take advantage of the elimination power of DEE without requiring its large computational run-times, while trading off a certain degree of optimality. In general, it does so by assuming a given rotamer assignment at all but one protein position and then exhaustively...

Design Strategy

The modular protein design strategy is highly versatile and can employ peptide sequences derived from wild-type proteins, computationally guided amino-acid sequences, and or peptide motifs discovered using high-throughput screening Figure 8.2 . While the majority of protein-based materials described to date have used wild-type amino-acid sequences, there are notable examples of engineered sequences as well. For example, high-throughput screening of peptides was used to determine the relative...

Experimental Approaches For Highthroughput Design Sampling

Despite recent advances, the success rate for computer-based protein designs is well below 100 . Therefore, it is important to be able to experimentally characterize as many designs as possible. Typically, this requires cloning the gene for the designed polypeptide in an expression vector to allow subsequent microbial expression and purification for in vitro characterization. This one-protein-at-a-time approach is expensive, tedious, and time consuming. There are two approaches that in the near...

The molecular scaffold concept

The idea of constructing a new molecular recognition function through the engineering of a section of protein surface i.e., a patch Figure 5.1A originates from the molecular architecture of immunoglobulins Figure 5.1B . The immune system can produce antibodies against virtually any type of antigen by, to a first-order approximation, simply tuning the amino-acid sequences of a total of six short segments termed complementarity determining regions CDRs . CDRs are surface-exposed loops located at...

Mapping binding energetics

Although most phage display studies have focused on either improving natural protein functions or developing novel protein functions, the method can also be used to understand the molecular basis of protein structure and function. Libraries with restricted diversity have proven to be highly effective for studying protein function, because reducing the chemical complexity of the library greatly simplifies the identification of key interactions within a binding interface Morrison and Weiss 2001...

Library screening methods

Cell surface display systems are valuable tools for protein engineering when coupled with a method for high-throughput screening of large libraries Boder and Wittrup 1998 Daugherty et al. 1998 . While flow cytometry remains a common method for screening cell surface display libraries, other methods such as magnetic bead sorting and panning have also been developed. The library screening strategies discussed in this section are generally applicable to multiple hosts and display formats....

Grasp

Public index.php Software GRASP html probe.php Structural visualization program Structural visualization program Visualization of protein surfaces and electrostatic potential maps GRASP scripts for computing the residual potential Software for computation of steric complementarity the backbone only. These cartoon models are useful for understanding the overall architecture of a protein or protein complex, and thus in identifying particular regions to include as variables in a combinatorial...

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Second generation 102 residues, a-helical, well folded Second generation 102 residues, a-helical, well folded FIGuRE 11.5 see color insert following page 178 Design of the first- and second-generation libraries of binary patterned four-helix bundles. The first generation library was designed to have 74 residues. In the second generation, each helix was extended by one helical turn leading to a 102-residue library. The design templates of the first- and second-generation four-helix bundle...

Conclusion and future prospects

Protein therapeutics are a very important part of modern medicine, and in certain situations they are the only effective therapies. Expanding with an ever-increasing speed, the protein therapeutics market is projected to reach 87 billion by year 2010 Kalorama Information 2006 . To realize such great potential, continuous efforts are required to optimize their efficacy, while simultaneously discovering novel protein drugs. As exemplified in this chapter, protein engineering and design have long...

Conclusion

Substantial progress has been made in the design of protein structure. Observations from natural proteins and mutagenesis experiments, as well as learning from failed protein design attempts, have guided our current understanding. As these examples illustrate, the iteration between computational design and experimental validation is crucial toward improving our models. The approximations necessary for speed in protein design come at the expense of accuracy, and methods for circumventing these...

Case studies

Elastin-like polypeptides have been widely studied as potential injectable biomaterials and implantable scaffolds for regenerative medicine and drug delivery Chilkoti et al. 2002 Rodriguez-Cabello et al. 2007 . Potential applications include cartilage and invertebral disk repair Betre et al. 2006, Betre et al. 2002 , small diameter vascular grafts Liu et al. 2004 , and spinal cord repair Straley and Heilshorn 2009 . Elastin motifs are attractive components of materials for tissue engineering...

Contributor List

Department of Bioengineering University of Pennsylvania Philadelphia, Pennsylvania Codon Devices, Inc. Cambridge, Massachusetts Codon Devices, Inc. Cambridge, Massachusetts Department of Chemistry Princeton University Princeton, New Jersey Biophysics University of North Carolina Chapel Hill, North Carolina Biophysics Graduate Group University of California Berkeley, California and Pharmaceutical Science University of California San Diego, California Department of Chemical Engineering...

Sheldon J Park Jennifer R Cochran

Taylor amp Francis Group, an informa business 6000 Broken Sound Parkway NW, Suite 300 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor amp Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number 978-1-4200-7658-5 Hardback This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made...

Roles Of Interface Topography

Monoclonal Antibody Paratope Epitop

Studies of natural interfaces have suggested that the interface topography is a major determinant of protein-protein interaction. The topography of the intended paratope the interface on the antibody binder side that interacts with the epitope is strongly influenced by the choice of scaffold surfaces to be used, and it may be a greater determinant of scaffold capacity than the secondary structure class. The overall shape of a paratope can be convex, flat, or concave. Furthermore, the convex and...

Designing Function

Designing protein function requires consideration of both stability and binding. The task is particularly challenging when designing enzymes. An enzyme not only must bind a target substrate, but also needs to stabilize the transition state and release the product Walsh 2001 . The geometry of catalytic residues must be positioned accurately to support efficiency catalysis. Designed enzymes include catalytic antibodies that are engineered by affinity maturation in the presence of a...

Design Of Preorganized Structures

Although the entropic cost of folding a single chain is less than that required for the intermolecular association of modules, the cost of forming a unique three-dimensional structure from an unconstrained linear chain is still substantial. The unfavorable con-formational entropy change associated with folding into a unique structure can be decreased by preorganizing the desired structure. Several groups have employed this strategy by using covalent cross-links, synthetic templates, or ligand...

Flow Cytometry

Flow cytometry allows for the rapid measurement of multiple optical properties of individual cells, particles, or compartmentalized droplets. Fluorescence-activated cell sorting FACS involves the flow cytometric separation of members of the cell population with desired fluorescent properties and is not limited only to cells . Modern FACS instruments are capable of sorting up to 109 cells in one or a few days. Note that these screening capabilities approach the limits of library cloning and...

Advances In Display Technologies

An important aspect of successful interface design and engineering is the ability to generate and test a large number of amino-acid sequence diversities. Thus, molecular display technologies are an indispensable tool in directed evolution-oriented interface engineering. Phage display Chapter 1 has been the most commonly used display technology in this field. However, other methods such as yeast display Chapter 2 , and ribosome display and mRNA display Chapter 3 have been successfully used. In...

The Binary Code Strategy For Protein Design

Protein Binary Code

The binary code strategy is based on the design of specific patterns of polar and nonpolar amino acids in the primary sequence. The strategy is described as binary because the amino acids are treated as belonging to one of two groups polar or nonpolar. This polar nonpolar patterning is designed to match the inherent structural periodicity of the desired secondary structure a-helix or -strand , and thereby specify amphiphilic units of secondary structure Figure 11.3 . When these amphiphilic...

Emerging Cell suRFACE DisPLAY systems INsEct Cell And Mammalian Cell Display

Insect cell baculovirus display is a well-established technology, yet unlike bacterial or yeast surface display, this system has not been extensively used for protein engineering applications Makela and Oker-Blom 2006 Makela and Oker-Blom 2008 . Despite the relatively few examples of protein engineering with insect cell baculo-virus systems, which require greater expertise in handling compared to microbial-based systems, recent studies have shown much potential. This format involves either the...

CELL suRFACE DisPLAY ouTLooK

Bacterial and yeast surface display are both well-established systems for protein engineering. Continued development of these methods will bring more goals within reach, such as engineering novel enzymes and integral membrane proteins. While currently less developed, insect cell and mammalian cell surface display have the potential to address the limitations of the more established platforms. Efforts that combine protein library screening with computational modeling and bioinformatics methods...