Patrik Forrer

Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.

Designed to be stable: Crystal structure of a consensus ankyrin repeat protein

Ankyrin repeat (AR) proteins mediate innumerable protein–protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-Å resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat–repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the β-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

In vitro display technologies: novel developments and applications

In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.

Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10(10) individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFalpha, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.

Novel fold and capsid-binding properties of the |[lambda]|-phage display platform protein gpD

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage λ, was solved at 1.1 Å resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0.132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 Å resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

Beyond binding: using phage display to select for structure, folding and enzymatic activity in proteins

Phage display of a wide range of polypeptides has been increasingly used to identify novel molecules with useful binding properties for research, medical and industrial applications. Recent developments include methods for the selection of stabilized variants of a protein, the selection of regulatable enzymes and promising strategies for the selection and evolution of protein catalysts.

Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family.

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.

A novel strategy to design binding molecules harnessing the modular nature of repeat proteins

Repeat proteins, such as ankyrin or leucine‐rich repeat proteins, are ubiquitous binding molecules, which occur, unlike antibodies, intra‐ and extracellularly. Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target‐binding surfaces. Based on this modularity, we developed a novel strategy to generate combinatorial libraries of polypeptides with highly diversified binding specificities. This strategy includes the consensus design of self‐compatible repeats displaying variable surface residues and their random assembly into repeat domains. We envision that such repeat protein libraries will be highly valuable sources for novel binding molecules especially suitable for intracellular applications.

Consensus design of repeat proteins.

Consensus design is a valuable protein‐engineering method that is based on statistical information derived from sequence alignments of homologous proteins. Recently, consensus design was adapted to repeat proteins. We discuss the potential of this novel repeat‐based approach for the design of consensus repeat proteins and repeat protein libraries and summarize recent results from such experiments.

Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications

The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.