S-layer-mediated display of the immunoglobulin G-binding domain of streptococcal protein G on the surface of Caulobacter crescentus: development of an immunoactive reagent

Abstract

The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative, Staphylococcus aureus protein A. Caulobacter crescentus produces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell as S. aureus cells (Pansorbin). The ability of recombinant protein G-Caulobacter cells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobacter cells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.

FIG. 1.

SDS-PAGE (A) and Western immunoblot (B) analyses of normalized low-pH-extracted RsaA-protein G peptide (GB1) display constructs. Equal volumes of protein preparations were loaded. Immunoblot were treated with HRP-conjugated goat anti-rabbit antibody (1:1,000) and developed with 4-chloro-1-naphthol. M, molecular mass standard (in kDa). Lanes: 1, p4ARsaA(723Δ); 2, p4ARsa(574)/GB1_XS; 3, p4ARsa(622)/GB1_XS; 4, p4ARsa(690)/GB1_XS; 5, p4ARsa(723)/GB1_XS; 6, p4ARsa(944)/GB1_XS.

FIG. 2.

Colloidal gold labeling of chimeric RsaA-protein G (GB1_XS) S-layer on the surface of C. crescentus cells. Whole cells expressing chimeric constructs RsaA(690)/GB1_XS (A) and RsaA(944)/GB1_XS (B) were incubated with normal rabbit antiserum followed by 5 nM protein A-colloidal gold labeling.

FIG. 3.

SDS-PAGE (A) and Western immunoblot (B) analyses of normalized low-pH-extracted RsaA and various recombinant RsaA proteins containing the original GB1_XS and sequential additions of GB1_SN and Muc1B sequences. Immunoblots were treated with HRP-conjugated goat anti-mouse antibody (1:5,000) and developed with 4-chloro-1-naphthol. M, molecular mass standard (in kDa). Lanes: 1, p4ARsaA(723Δ); 2, p4ARsaA(723Δ)/GB1_XS; 3, p4ARsaA(723Δ)/GB1_SN; 4, p4ARsaA(723Δ)/GM; 5, p4ARsaA(723Δ)/GMG; 6, p4ARsaA(723Δ)/GMGMG; 7, p4ARsaA(723Δ)/MGMGMGM.

FIG. 4.

Immunoprecipitation of FLAG-tagged protein from eukaryotic cell lysates by using anti-FLAG antibody and protein G beads or Caulobacter cells expressing RsaA(723Δ) or recombinant RsaA(723Δ)/MGMGMGM. Molecular mass standards are indicated in kDa. The arrow indicates the position of the FLAG-tagged RapGapII protein. The reactive proteins of smaller size in lanes 3 and 5 are Ig heavy chains, and the bands above the arrow reflect antibody binding to RsaA(723Δ)/MGMGMGM.