Per-Åke Nygren

Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold

In recent years, classical antibody‐based affinity reagents have been challenged by novel types of binding proteins developed by combinatorial protein engineering principles. One of these classes of binding proteins of non‐Ig origin are the so‐called affibody binding proteins, functionally selected from libraries of a small (6 kDa), non‐cysteine three‐helix bundle domain used as a scaffold. During the first 10 years since they were first described, high‐affinity affibody binding proteins have been selected towards a large number of targets for use in a variety of applications, such as bioseparation, diagnostics, functional inhibition, viral targeting and in vivo tumor imaging/therapy. The small size offers the possibility to produce functional affibody binding proteins also by chemical synthesis production routes, which has been found to be advantageous for the site‐specific introduction of various labels and radionuclide chelators.

Consequences of Membrane Protein Overexpression in Escherichia coli

Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Scaffolds for engineering novel binding sites in proteins

The combination of combinational protein chemistry and powerful selection techniques has resulted in the development of novel protein ligands based on the randomization of surface residues of a parental protein which is used as a scaffold. Such binding proteins, selected from libraries via specific binding towards a given target ligand, have the potential to replace natural antibodies in various biotechnological applications.

Engineering proteins to facilitate bioprocessing

Genetic engineering is now being applied to aid the purification of recombinant proteins. The addition of specifically designed tags or the modification of sequences within the target-gene product has enabled the development of novel strategies for downstream processing that can be employed for efficient recovery of both native or modified proteins. This article discusses novel trends in genetic engineering that aid the bioprocessing of recombinant proteins.

Affibody Molecules in Biotechnological and Medical Applications

Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.

The serum albumin-binding region of streptococcal protein G: a bacterial fusion partner with carrier-related properties.

In this study, we have explored the use of the serum albumin-binding region (BB) from streptococcal protein G (SpG) as a bacterial fusion partner for production of peptide immunogens. The fusion protein BB-M3, containing BB and repeated structures from the Plasmodium falciparum malaria antigen Pf155/RESA, was efficiently purified from Escherichia coli culture supernatants by affinity chromatography using BB as an affinity tag. Rabbits immunized with BB-M3 in Freunds adjuvant produced high levels of antibodies which reacted with both M3 and BB in ELISA and stained intact Pf155/RESA in the membrane of infected erythrocytes. These antibody levels were sustained for more than 30 weeks. BB-M3 also induced antibody responses to M3, BB and intact Pf155/RESA in a number of mouse strains, including several strains which are non-responders to the malaria sequences. In the latter mice, however, BB-M3 only activated BB-specific T cells, suggesting that BB has ability to provide carrier-related T cell help for antibody production. Moreover, the minimal albumin-binding motif of SpG, containing only 46 amino acids, was immunogenic in both B10.BR, B10.D2 and C57BL/6 mice (H-2k, H-2d and H-2b, respectively). These results indicate that BB has both affinity tag and carrier-related properties and suggest that fusion proteins containing BB can be efficient tools for the generation of antibody responses to peptides which are weak immunogens.

An affibody in complex with a target protein: Structure and coupled folding

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The ZSPA-1 affibody has been selected from a phage-displayed library as a binder to protein A. ZSPA-1 also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the ZSPA-1 affibody in its uncomplexed state and determined the solution structure of a Z:ZSPA-1 protein–protein complex. Uncomplexed ZSPA-1 behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on ZSPA-1. We also note similarities in how the surface of the Z domain responds by induced fit to binding of ZSPA-1 and Ig Fc, respectively, suggesting that the ZSPA-1 affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

Engineering of Fc1 and Fc3 from human immunoglobulin G to analyse subclass specificity for staphylococcal protein A

A system for production of recombinant Fc fragments of human IgG in Escherichia coli has been developed to allow for structural and functional studies of human Fc. The genes for the Fc fragments of human IgG subclasses 1 and 3, designated Fc(1) and Fc(3), were cloned from a human spleen cDNA library. The interactions to Staphylococcal protein A (SpA), a bacterial Fc receptor, that interacts with human IgG-Fc(1), but not with human IgG-Fc(3), were analyzed. To corroborate the involvement of amino acid residues in Fc, responsible for these differences in binding, two Fc variants were constructed; Fc(1(3)) and Fc(3(1)), each containing an isotypic dipeptide substitution. Production levels in E. coli of 1-10 mg/l of secreted Fc proteins, covalently linked as dimers, were routinely obtained. SpA-binding analyses of all four Fc variants using biosensor technology, showed that Fc(1) and Fc(3(1)) interact with SpA, while Fc(3) and Fc(1(3)) lack detectable SpA binding. The rendered SpA binding of the Fc variant Fc(3(1)), is concluded to result from the introduced dipeptide substitution (R435H, F436Y). The results demonstrate that the Fc expression system efficiently can be used in Fc engineering.

Structural basis for recognition by an in vitro evolved affibody.

The broad binding repertoire of antibodies has permitted their use in a wide range of applications. However, some uses of antibodies are precluded due to limitations in the efficiency of antibody generation. In vitro evolved binding proteins, selected from combinatorial libraries generated around various alternative structural scaffolds, are promising alternatives to antibodies. We have solved the crystal structure of a complex of an all α-helical in vitro selected binding protein (affibody) bound to protein Z, an IgG Fc-binding domain derived from staphylococcal protein A. The structure of the complex reveals an extended and complementary binding surface with similar properties to protein–antibody interactions. The surface region of protein Z recognized by the affibody is strikingly similar to the one used for IgG1 Fc binding, suggesting that this surface contains potential hot-spots for binding. The implications of the selected affibody binding-mode for its application as a universal binding protein are discussed.