Application of Affitins for Affinity Purification of Proteins.

Affinity chromatography is a powerful purification technique, as it allows proteins of interest to be obtained at a high degree of purity in a single step. This technique can be applied on a research laboratory scale as well as on an industrial scale. The interaction involved in affinity separation most often involves a natural ligand or an antibody specific for the protein of interest, or the recognition of a peptide tag artificially added to the recombinant protein. Unfortunately, natural ligands are not always available and it may be undesirable or impossible to add a purification tag, especially for the production of therapeutic proteins. We have developed Affitins as a new class of artificial affinity proteins that can be generated against virtually any protein of interest. Due to their very high selectivity, their remarkable robustness against extreme acid or alkaline conditions and their low production cost, Affitins are particularly suited to this technique. We describe here the production of Affitins and their immobilization on resin beads to prepare affinity chromatography columns. The protocol also describes the use of these columns.

Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins.

Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.

Multivalent Affidendrons with High Affinity and Specificity toward Staphylococcus aureus as Versatile Tools for Modulating Multicellular Behaviors.

Multivalency is a widely occurring natural phenomenon often exploited in nanotechnology to enhance biorecognition. We report the preparation and characterization of versatile, multivalent Affitin-dendrimer conjugates (Affidendrons) showcased by a set targeting Staphylococcus aureus ( S. aureus), an opportunistic pathogen causing numerous hospital- and community-acquired infections. Affitins are small affinity proteins characterized by higher stability and lower cost-effective production than antibodies. The strategy presented provides a platform for the rational design of multivalent nanodevices that, retaining the ability of Affitins to recognize their target with high specificity, achieve a largely enhanced affinity. Affidendrons with precisely designed size and valency have been exploited to modulate complex multicellular behaviors of S. aureus, such as agglutination and biofilm formation. Agglutination assays showed that Affidendrons rapidly cross-link S. aureus strains with high bacterial cell selectivity. Moreover, remarkably low concentrations of Affidendrons were able to effectively prevent biofilm formation. Overall, Affidendrons represent a promising platform for the rapid and selective pathogen identification, infection imaging, and theranostic applications.

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications.

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

The archaeal "7 kDa DNA-binding" proteins: extended characterization of an old gifted family.

The "7 kDa DNA-binding" family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 µM to 22 µM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative "7 kDa DNA-binding" family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents.

Affitins for protein purification by affinity magnetic fishing.

Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing.

Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

Artificial affinity proteins as ligands of immunoglobulins.

A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized.

Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability.

As a useful reagent for biotechnological applications, a scaffold protein needs to be as stable as possible to ensure longer lifetimes. We have developed archaeal extremophilic proteins from the "7 kDa DNA-binding" family as scaffolds to derive affinity proteins (Affitins). In this study, we evaluated a rational structure/sequence-guided approach to stabilize an Affitin derived from Sac7d by transferring its human IgG binding site onto the framework of the more thermally stable Sso7d homolog. The chimera obtained was functional, well expressed in Escherichia coli, but less thermally stable than the original Affitin (T(m) = 74.2 °C vs. T(m) = 80.4 °C). Two single mutations described as thermally stabilizing wild type Sso7d were introduced into chimeras. Only the double mutation nearly restored thermal stability (T(m) = 76.9 °C). Interestingly, the chimera and its double mutant were stable from pH 0 up to at least pH 13. Our results show that it is possible to increase further the stability of Affitins toward alkaline conditions (+2 pH units) while conserving their advantageous properties. As Affitins are based on a growing family of homologs from archaeal extremophiles, we conclude that this approach offers new potential for their improvement, which will be useful in demanding biotechnological applications.

Affinity transfer to the archaeal extremophilic Sac7d protein by insertion of a CDR.

Artificially transforming a scaffold protein into binders often consists of introducing diversity into its natural binding region by directed mutagenesis. We have previously developed the archaeal extremophilic Sac7d protein as a scaffold to derive affinity reagents (Affitins) by randomization of only a flat surface, or a flat surface and two short loops with natural lengths. Short loops are believed to contribute to stability of extremophilic proteins, and loop extension has been reported detrimental for the thermal and chemical stabilities of mesophilic proteins. In this work, we wanted to evaluate the possibility of designing target-binding proteins based on Sac7d by using a complementary determining region (CDR). To this aim, we inserted into three different loops a 10 residues CDR from the cAb-Lys3 anti-lysozyme camel antibody. The chimeras obtained were as stable as wild-type (WT) Sac7d at extreme pH and their structural integrity was supported. Chimeras were thermally stable, but with T(m)s from 60.9 to 66.3°C (cf. 91°C for Sac7d) which shows that loop extension is detrimental for thermal stability of Sac7d. The loop 3 enabled anti-lysozyme activity. These results pave the way for the use of CDR(s) from antibodies and/or extended randomized loop(s) to increase the potential of binding of Affitins.

Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Tolerance of the archaeal Sac7d scaffold protein to alternative library designs: characterization of anti-immunoglobulin G Affitins.

Engineered protein scaffolds have received considerable attention as alternatives to antibodies in both basic and applied research, as they can offer superior biophysical properties often associated with a simpler molecular organization. Sac7d has been demonstrated as an effective scaffold for molecular recognition. Here, we used the initial L1 'flat surface' library constructed by randomization of 14 residues, to identify ligands specific for human immunoglobulin G. To challenge the plasticity of the Sac7d protein scaffold, we designed the alternative L2 'flat surface & loops' library whereof only 10 residues are randomized. Representative binders (Affitins) of the two libraries exhibited affinities in the low nanomolar range and were able to recognize different epitopes within human immunoglobulin G. These Affitins were stable up to pH 12 while largely conserving other favorable properties of Sac7d protein, such as high expression yields in Escherichia coli, solubility, thermal stability up to 80.7°C, and acidic stability (pH 0). In agreement with our library designs, mutagenesis study revealed two distinct binding areas, one including loops. Together, our results indicate that the Sac7d scaffold tolerates alternative library designs, which further expands the diversity of Affitins and may provide a general way to create tailored affinity tools for demanding applications.

Ribosome display for the selection of Sac7d scaffolds.

Combinatorial libraries of Sac7d have proved to be a valuable source of proteins with favorable biophysical properties and novel ligand specificities, so-called Nanofitins. Thus, Sac7d represents a promising scaffold alternative to antibodies for biotechnological and potentially clinical applications. We describe here the methodology for the construction of a library of Sac7d and its use for selection by ribosome display.

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD.

We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf.

BACKGROUND: Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. RESULTS: Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7). CONCLUSION: Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

Identification of functionally important residues in the pyridoxal-5'-phosphate-dependent catalytic antibody 15A9.

Antibody 15A9 is unique in its ability to catalyze the transamination reaction of hydrophobic D-amino acids with pyridoxal-5'-phosphate (PLP). Both previous chemical modification studies and a three dimensional (3-D) homology model indicated the presence of functionally important tyrosine residues in the antigen-binding cavity of antibody 15A9. To gain further insight into the hapten, ligand binding, and catalytic mechanism of 15A9, all tyrosine residues in the complementarity-determining regions (CDRs) and the single arginine residue in CDR3 of the light chain were subject to an alanine scan. Substitution of Tyr(H33), Tyr(L94), or Arg(L91) abolished the catalytic activity and reduced the affinity for PLP and N(a)-(5'-phosphopyridoxyl)-amino acids, which are close analogues of covalent PLP-substrate adducts. The Tyr(H100b)Ala mutant possessed no detectable catalytic activity, while its affinity for each ligand was essentially the same as that of the wild-type antibody. The binding affinity for the hapten was drastically reduced by a Tyr(L32)Ala mutation, suggesting that the hydroxyphenyl group of Tyr(L32) participates in the binding of the extended side chain of the hapten. The other Tyr --> Ala substitutions affected both binding and catalytic activity only to a minor degree. On the basis of the information obtained from the mutagenesis study, we docked N(alpha)-(5'-phosphopyridoxyl)-D-alanine into the antigen-binding site. According to this model, Arg(L91) binds the alpha-carboxylate group of the amino acid substrate and Tyr(H100b) plays an essential role in the catalytic mechanism of antibody 15A9 by facilitating the Calpha/C4' prototropic shift. In addition, the catalytic apparatus of antibody 15A9 revealed several mechanistic features that overlap with those of PLP-dependent enzymes.

Development of nonradioactive microtiter plate assays for nuclease activity.

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.

Pyridoxal-5'-phosphate-dependent catalytic antibodies.

Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5'-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N(alpha)-(5'-phosphopyridoxyl)-L-lysine, a stable analog of the covalent coenzyme-substrate adducts were screened by a competition ELISA for binding of the PLP-amino acid Schiff base adduct. The Schiff base with its C4'-N alpha double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent alpha,beta-elimination reaction of beta-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k(cat)'=0.42 min(-1) with D-alanine at 25 degrees C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme-amino acid adduct. (2) The bond at C alpha to be broken lies together with the C alpha-N bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The alpha-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme-substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10(4) times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k(cat (Ab x PLP))'/k(cat (PLP))'=5 x 10(3)). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.

A method for the detection and screening of catalytic anti-DNA antibodies.

We have developed a microtiter plate assay for the detection and screening of anti-DNA hydrolytic antibodies. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5'-end of the 3'-biotinylated DNA strands. The substrate binds specifically to the wells of streptavidin-coated microtiter plates where the reaction takes place. Uncleaved substrate retains the digoxigenin label, which is then detected with an enzyme-labeled anti-digoxigenin antibody. We first assessed the efficiency of this assay by measuring S1 nuclease and DNase I activities and the inhibitory effect of EDTA on the reaction. The ALONA procedure was then successfully applied to the screening of a high number of hybridoma clones derived from nonimmunized (NZB x NZW)F1 mice with spontaneous lupus erythematosus. We detected three potential catalytic antibodies and investigated their substrate specificity. Overall, our findings demonstrate the value of the ALONA method for high throughput screening of potential nucleases and catalytic antibodies. Although this assay was designed for the selection of catalysts active in DNA hydrolysis, it can be adapted to detect most types of substrate cleavage reaction.