Engineering sperm-binding IgG antibodies for the development of an effective nonhormonal female contraception.

Many women risk unintended pregnancy because of medical contraindications or dissatisfaction with contraceptive methods, including real and perceived side effects associated with the use of exogenous hormones. We pursued direct vaginal delivery of sperm-binding monoclonal antibodies (mAbs) that can limit progressive sperm motility in the female reproductive tract as a strategy for effective nonhormonal contraception. Here, motivated by the greater agglutination potencies of polyvalent immunoglobulins but the bioprocessing ease and stability of immunoglobulin G (IgG), we engineered a panel of sperm-binding IgGs with 6 to 10 antigen-binding fragments (Fabs), isolated from a healthy immune-infertile woman against a unique surface antigen universally present on human sperm. These highly multivalent IgGs (HM-IgGs) were at least 10- to 16-fold more potent and faster at agglutinating sperm than the parent IgG while preserving the crystallizable fragment (Fc) of IgG that mediates trapping of individual spermatozoa in mucus. The increased potencies translated into effective (>99.9%) reduction of progressively motile sperm in the sheep vagina using as little as 33 µg of the 10-Fab HM-IgG. HM-IgGs were produced at comparable yields and had identical thermal stability to the parent IgG, with greater homogeneity. HM-IgGs represent not only promising biologics for nonhormonal contraception but also a promising platform for engineering potent multivalent mAbs for other biomedical applications.

Simultaneous Soluble Secretion and Surface Display of Proteins in Saccharomyces cerevisiae Using Inefficient Ribosomal Skipping.

Combinatorial library screening platforms, such as yeast surface display, typically identify several candidate proteins that need further characterization and validation using soluble recombinant protein. However, recombinant production of these candidate proteins involves tedious and time-consuming subcloning steps. This, in turn, limits the number of candidate proteins that can be characterized. To address this bottleneck, we have developed a platform that exploits inefficient ribosomal skipping by the F2A peptide for simultaneous soluble secretion and cell surface display of protein in the yeast Saccharomyces cerevisiae. Here we provide detailed protocols utilizing this F2A-based yeast display system. We discuss specific recommendations for the purification of the secreted protein. Additionally, we provide suggestions for testing the functionality and binding specificity of the soluble secreted proteins using flow cytometry analysis.

An Engineered Sso7d Variant Enables Efficient Magnetization of Yeast Cells.

Magnetization using cheap and minimally toxic materials, such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here, we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein-but not other Sso7d mutants with similar overall protein charge or amino acid composition-preferentially bind iron oxide when present in a solution with high protein concentration and in the presence of 1000-fold excess of competitor yeast cells. Moreover, coexpression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications, where easy separation of cells or organelles from complex media is desirable.

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae.

The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.

Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.

Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme.

We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting nonoverlapping epitopes; individual binders of KD ∼ 1.3 µM and 250 nM produced a bivalent binder with apparent KD ∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (TM = 84.5 °C) and high recombinant expression yields in E. coli. Finally, when binders comprising the bivalent protein are fused to two of the three fragments of tripartite split-green fluorescent protein (GFP), target-dependent reconstitution of fluorescence occurs, thereby enabling a "mix-and-read" assay for target quantification.