Part:BBa_K4130001
IgG F(c) Binding Protein, EibD
Escherichia coli Ig-binding protein D (EibD) is an outer-membrane protein capable of non-immunological binding of IgG and IgA.
Biology
E. coli Ig-binding proteins (Eibs) are a wide-spread class of proteins within intimin-negative, shiga-toxin producing enterohemorrhagic E. coli (EHEC) strains [1]. First discovered in strain ECOR-9 [2, 3], the class has grown to include EibA, C, D, E, F, and G. The class is united in its common characteristic of immunoglobulin (Ig) binding activity.
Immunoglobulins, also known as antibodies, are a family of heterodimeric proteins produced by B cells capable of binding with great specificity to respective antigens. Igs are typically composed of a “variable region”, responsible for binding of the antigen, and a “constant region” with no affinity for the antigen. Igs are further classified according to their “constant regions”, into 5 classes: IgM, IgG, IgA, IgD, and IgE. [4]
E. coli Ig-binding proteins have been shown to be capable of binding the constant region of assorted Ig classes. Affinity and specificity for different classes of Ig proteins varies between Eib family members, with dissociation constants on the 100nM scale [5].
EibD (from E. coli strain ECOR-9) is a 511 amino acid, 210kDa [3] member of the Eib family. The protein exhibits affinity toward both IgG (Kd = ~73nM) and IgA (Kd = ~135nM) [5], with separate binding domains for each Ig [6]. The structure also includes a membrane anchor, left-handed coiled-coil, saddle domain, right-handed coiled-coil, neck, and head domain [6], characteristic of trimeric autotransporter adhesins - a family of secreted proteins in Gram-negative bacteria that are associated with virulence [7]. Besides Ig-binding qualities, heterologous expression of EibD has resulted in self-aggregation phenotypes [6], typical of other trimeric autotransporter adhesins such as YadA [7]. Transmission electron microscopy (TEM) imaging has revealed zipper-like structures forming between neighboring bacteria harboring EibD, explaining the autoaggregation phenotype. Although the biological function of EibD is unknown, its Ig-binding properties and homology to other autotransporter adhesins implicates its role in serum resistance. Binding of IgG by EibD may block binding of immunoglobulins to the adaptive immunity protein C1q, protecting bacteria against innate host defenses [6].
Part Uses
Given the Ig-binding properties of EibD, we chose to exploit it for whole-cell agglutination/aggregation assays to detect small molecules and proteins. Expression of EibD results in auto-agglutination and biofilm formation that can be observed via the eye. By incubating EibD-expressing bacteria with immunoglobulin antibodies, we hoped to be able to detect various antigens. Introduction of the antibodies would cause visual and quantitative dis-agglutination by molecular competition between self-self interactions and self-antibody interactions. Addition of the antigen would lead to bivalent binding interactions, causing re-agglutination of the cells.
Uniquely, this strategy of detection has the potential to be applied to nearly any antigen with zero need for further genetic engineering. Previous whole-cell bacterial sensors have been focused on expressing antibodies or antibody fragments on the surface of the bacterium, meaning that each strain is specific to an antigen. By expressing a general antibody-binding protein, a single strain can be utilized for the detection of multiple antigens.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1348
- 1000COMPATIBLE WITH RFC[1000]
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