scholarly journals Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1

1995 ◽  
Vol 310 (1) ◽  
pp. 177-184 ◽  
Author(s):  
K N Walker ◽  
S P Bottomley ◽  
A G Popplewell ◽  
B J Sutton ◽  
M G Gore
Keyword(s):  
Protein A ◽  
Spectroscopic Studies ◽  
Binding Domain ◽  
Protein G ◽  
Binding Interaction ◽  
Fc Fragment ◽  
Igg Binding ◽  
Immunoglobulin Binding Protein ◽  
Human Igg1 ◽  
Immunoglobulin Binding

A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.

scholarly journals 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

2007 ◽  
Vol 73 (10) ◽  
pp. 3245-3253 ◽  
Author(s):  
John F. Nomellini ◽  
Gillian Duncan ◽  
Irene R. Dorocicz ◽  
John Smit
Keyword(s):  
Immunoglobulin G ◽  
Tandem Repeats ◽  
Caulobacter Crescentus ◽  
Protein A ◽  
Cost Effective ◽  
Binding Domain ◽  
Protein G ◽  
Wild Type ◽  
Igg Binding ◽  
Streptococcal Protein G

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.

scholarly journals Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus

2003 ◽  
Vol 31 (3) ◽  
pp. 716-718 ◽  
Author(s):  
N.G. Housden ◽  
S. Harrison ◽  
S.E. Roberts ◽  
J.A. Beckingham ◽  
M. Graille ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein A ◽  
Cell Wall Protein ◽  
Protein G ◽  
Protein L ◽  
Heavy Chains ◽  
Binding Domains ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

Protein L is a multidomain cell-wall protein isolated from Peptostreptococcus magnus. It belongs to a group of proteins that contain repeated domains that are able to bind to Igs without stimulating an immune response, the most characterized of this group being Protein A (Staphylococcus aureus) and Protein G (Streptococcus). Both of these proteins bind predominantly to the interface of CH2-CH3 heavy chains, while Protein L binds exclusively to the VL domain of the κ-chain. The function of these proteins in vivo is not clear but it is thought that they enable the bacteria to evade the host's immune system. Two binding sites for κ-chain on a single Ig-binding domain from Protein L have recently been reported and we give evidence that one site has a 25–55-fold higher affinity for κ-chain than the second site.

Kinetics of the Antibody Recognition Site in the Third IgG-Binding Domain of Protein G

2016 ◽  
Vol 128 (33) ◽  
pp. 9719-9722 ◽  
Author(s):  
Supriya Pratihar ◽  
T. Michael Sabo ◽  
David Ban ◽  
R. Bryn Fenwick ◽  
Stefan Becker ◽  
...  
Keyword(s):  
Binding Domain ◽  
Recognition Site ◽  
Protein G ◽  
Antibody Recognition ◽  
The Third ◽  
Igg Binding ◽  
Kinetics Of

The Third IgG-Binding Domain from Streptococcal Protein G

1994 ◽  
Vol 243 (5) ◽  
pp. 906-918 ◽  
Author(s):  
Jeremy P. Derrick ◽  
Dale B. Wigley
Keyword(s):  
Binding Domain ◽  
Protein G ◽  
The Third ◽  
Igg Binding ◽  
Streptococcal Protein G

Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes

1992 ◽  
Vol 101 (2) ◽  
pp. 315-322 ◽  
Author(s):  
M. Terasaki ◽  
T.S. Reese
Keyword(s):  
Endoplasmic Reticulum ◽  
Cultured Cells ◽  
Protein A ◽  
Epithelial Cell Line ◽  
Cultured Fibroblasts ◽  
Whole Cells ◽  
Membrane Compartments ◽  
Immunoglobulin Binding Protein ◽  
Immunoglobulin Binding ◽  
Peripheral Regions

The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiO6(3) in CV-1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG

1998 ◽  
Vol 10 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Nicola L. Brown ◽  
Stephen P. Bottomley ◽  
Michael D. Scawen ◽  
Michael G. Gore
Keyword(s):  
Protein A ◽  
Binding Domain ◽  
Staphylococcal Protein A ◽  
Staphylococcal Protein ◽  
Igg Binding ◽  
Rabbit Igg

Kinetics of the Antibody Recognition Site in the Third IgG-Binding Domain of Protein G

2016 ◽  
Vol 55 (33) ◽  
pp. 9567-9570 ◽  
Author(s):  
Supriya Pratihar ◽  
T. Michael Sabo ◽  
David Ban ◽  
R. Bryn Fenwick ◽  
Stefan Becker ◽  
...  
Keyword(s):  
Binding Domain ◽  
Recognition Site ◽  
Protein G ◽  
Antibody Recognition ◽  
The Third ◽  
Igg Binding ◽  
Kinetics Of
Sign in / Sign up

Export Citation Format

Share Document