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.
•Affitins are extremophilic artificial affinity proteins.•We evaluated Affitins for their use as tailored reagents for affinity purification.•We immobilized anti-PulD, anti-HEWL and anti-hIgG Afftins ...on agarose matrix.•The columns obtained showed exquisite selectivity and a high robustness to CIP.•Affitins are thus a new class of on demand reagents for affinity chromatography.
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 0.25M 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.
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.
•Affitins are extremophilic affinity proteins derived from the Sac7d family.•We evaluated a rational approach to stabilize a Sac7d derived anti-IgG Affitin.•We grafted its IgG binding site onto the ...framework of the more stable Sso7d homolog.•We obtained a highly stable anti-IgG Affitin: Tm=76.9°C, pH 0 up to at least pH 13.•We thus further expand potential of Affitins to harsher biotechnological applications.
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 “7kDa 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 (Tm=74.2°C vs. Tm=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 (Tm=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.
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.
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.
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).
Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d polypeptides belonging to the archaeal Sul7d family. Recently, we have characterized this family and identified Aho7c as its smallest member. Here, Aho7c‐based Affitins specific for EpCAM were obtained after selection by ribosome display. They showed high stability (up to 74°C; pH 0–12) and affinities (KD = 110 pM), and thus Aho7c is proposed as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d‐based ones.
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.
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 Tms 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.
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.
Affitins 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.
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