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Investigation of affinity properties of receptor protein G of group G streptococci in different flow-through models using the approaches of affinity high-performance monolithic disk chromatography

Abstract number: P1313

Lozhkina O., Gupalova T., Totolian A., Tennikova T.

Objectives: The in vitro study of surface proteins of pathogenic microorganisms in model systems imitating its natural microsurrounding allows to estimate the role of receptors in pathogenesis of bacteria. The aim of the present study was to develop several flow-through models with cell-imitators, intact and inactivated cells of Group G streptococci immobilised on CIM® monolithic stationary phases (BIA Separations, Slovenia) and to investigate them using the approaches of affinity high performance monolithic disk chromatography (AHPMDC) taking as a reference the interaction between surface protein G and human IgG, thoroughly studied before [1].

Methods: We developed and investigated four models of surface of Streptococcus: 1) cell-imitators on the base of monodisperse polymethylmethacrylate (PMMA) microspheres (1 mkm in diameter) with covalently attached recombinant protein G; 2) cell-imitators on the base of monodisperse PMMA microspheres functionalised with recombinant protein G and dextran, imitating polysaccharidic microsurrounding of the receptor protein, 3) inactivated cells of Group G streptococci (strain 148); 4) intact cells of Group G streptococci (strain 148). All models were covalently immobilised on the surface of flow-through pores of CIM® disks in order to examine such an affinity parameter as dissociation constant (Kdiss) of the interaction between protein G and human IgG by means of AHPMDC.

Results: Cell-imitators as well as cells themselves were immobilised on the surface of absolutely flow-through pores of monolithic sorbents and did not block them. Such a design of models allowed to realise the processes of elution at high speed (2.5 ml/min) in comparison to traditional affinity chromatography where average speed is about 1–2 ml/h. Kdiss evaluated for all the models described above were of the same magnitude, ranging around 10–6–10–7 M. These results are in good agreement with previously received data [1] and indicate that streptococcal protein G has an extremely high affinity to human IgG that does not depend on its microsurrounding and is not influenced by covalent immobilisation.

Conclusion: The developed methodical approach seems to be an attractive tool to study the interactions between human proteins and microorganisms in order to gain improved knowledge about the pathogenesis of the last ones.

References.

References

1. Lozhkina O.V., Gupalova T.V., Palagnuk V.G., Totolian A.A., Tennikova T.B. J. Chromatogr. A. – 2002, v. 949, p. 185.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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