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Applied and Environmental Microbiology, May 2007, p. 3245-3253, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02900-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

John F. Nomellini, Gillian Duncan, Irene R. Dorocicz,{dagger} and John Smit*

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

Received 14 December 2006/ Accepted 16 March 2007

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, 2509-2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3. Phone: (604) 822-4417. Fax: (604) 822-6041. E-mail: jsmit{at}interchange.ubc.ca

{triangledown} Published ahead of print on 23 March 2007.

{dagger} Present address: Clinical Genetics Technology, British Columbia Institute of Technology, Burnaby, BC, Canada.

Applied and Environmental Microbiology, May 2007, p. 3245-3253, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02900-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Smit, J., Kaltashov, I. A., Cotter, R. J., Vinogradov, E., Perry, M. B., Haider, H., Qureshi, N. (2008). Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus. Innate Immunity 14: 25-36 [Abstract]  


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