Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

International audienceAffitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications

Evolution of interleukin-15 for higher E. coli expression and solubility

International audienceDirected evolution was used to generate IL-15 mutants with increased solubility and cytoplasmic over-expression in Escherichia coli. A protein solubility selection method was used in which the IL-15 gene was expressed as an N-terminal fusion to chloramphenicol acetyltransferase (CAT) as reporter protein. Clones that grew in the presence of high concentrations of chloramphenicol were then screened by ELISA to assay the binding activity of the IL-15-CAT fusion to the IL-15Ra Sushi domain. Two variants of IL-15, M38 and M253, containing five mutations and one mutation respectively, were selected with a dramatic improvement in solubility; the soluble concentration in cell culture was 12-to 18-fold higher, respectively, than for WT IL-15. Characterization of their binding to IL-15Ra and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15Ra and acts as an effective agonist of IL-15

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

International audienceDetection 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

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

International audienceThe " 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. In living organisms, the long genomic DNA has to be packed in order to fit into cells, while the genetic information must stay accessible for replication and transcription events. To this aim, organisms have developed different compaction systems, such as the wrapping of DNA around histones to form the chromatin in Eukarya, and the supercoiling of DNA with the help of non-histone proteins to form the nucleoid in Bacteria. Archaea often live in extreme environments and have the additional challenge to protect their genomic DNA from extreme conditions, such as high temperatures. Many Archaea contain homologs of eukaryotic histones, but Desulfurococcales, Thermoplasmatales and Sulfolobales use a different kind of packaging proteins 1,2. Hyperthermophile and acidophile archaea of the Sulfolobales order from the Crenarchaeota kingdom express small basic DNA-binding proteins, which represent about 5% of the total soluble cellular proteins, sufficient to coat the entire genome of a Sulfolobus cell 3. These proteins constitute the family called " 7 kDa DNA-binding " or Sul7d 4. They were first isolated from Sulfolobus acido-caldarius which produces five of them, named Sac7a, b, c, d, and e. Sac7d and Sac7e are encoded by distinct genes, while Sac7a and b are truncated versions of Sac7d 5–7. Highly similar homologs have been found in all Sulfolobus species, such as Sso7d from Sulfolobus solfataricus 8 , and Ssh7a and Ssh7b from Sulfolobus shibatae-two proteins encoded by two distinct genes 3,9. Sac7d and Sso7d are the two most studied proteins of this family. They have been characterized for their structure, function, chemical stability and biophysical properties 7. Sac7d and Sso7d are hyperthermostable (T m = 90.4 °C and 100.2 °C, respectively) 10,11 and are resistant from pH 0 up to at least pH 12 12,13. Although Sac7d and Sso7d sequences show only few differences, Sso7d is more stable than Sac7d. Their three-dimensional structures show that they both fold as an SH3-like domain capped by a C-terminal α-helix 14,15 and that they sharply kink the double DNA helix upon binding into the minor groove 16,17. It has been shown that Sac7d and Sso7d are general dsDNA binders with K D values varying in a salt dependent manner from 20 nM (low salt) to 3.8 μ M (high salt) for Sac7d, and from 116 nM to 12.8 μ M for Sso7d, and with a preference for G/C rich sequences 18,19. Sac7d has the property to increase the thermal stability of DNA duplexes by as much as 43.5 °C 6,15. Furthermore, Ssh7a and Ssh7b have been partially characterized and an affinity for dsDNA of about 100 n

Affitins for protein purification by affinity magnetic fishing

International audienceCurrently 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 " 7 kDa 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 58 mg lysozyme/g support and 165 mg IgG/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

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)

Isolation and characterization of anti-FcgRIII (CD16) llama single-domain antibodies that activate natural killer cells

International audienceFcgRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific anti-bodies (bsAbs) targeting FcgRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcgRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgRIIIB extra-cellular domains. These sdAbs bind FcgRIIIA 1 NK cells and FcgRIIIB 1 poly-morphonuclear cells, but not FcgRI 1 or FcgRII 1 cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcgRIII with a K D in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-g production, respectively. These anti-FcgRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgRIII killer cells to target and destroy tumor cells

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

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

International audienceAffinity 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