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Bifunctional genetically engineered conjugates based on protein A of Staphylococcus aureus and their applications for affinity chromatography and immunodiagnostics


Presented Institute of Genetic and Regenerative Medicine of the National Academy of Medical Sciences of Ukrain

Ph.D., S.Sc., Head of the Laboratory of Genetic Engineering Biotechnology Institute of Genetic and Regenerative Medicine of the National Academy of Medical Sciences of Ukraine" Gorbatiuk Oksana Borysivna

SPA is a cell wall associated protein exposed on the surface of the S. aureus. SPA has high affinity to IgG from various animal species and human. The most applied affinity system for the purification of antibodies is based on SPA. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in E. coli system in a soluble form. The SPA-CBD2 fusion protein was affinity-immobilized on the microcrystalline cellulose CC31 and was used for purification of antibodies. The purity of antibodies in eluted fractions was more than 95 %. The designed bioaffinity sorbent provides obtaining pure poly and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse immunoglobulin G. Introduced C-terminal cysteine provided oriented covalent immobilization of SPA on maleimide-activated matrix.

A successful immobilization of SPA-Cys on a gold surface of the SPR spectrometer while preserving its high immunoglobulin-binding activity, selectivity and stability of the sensor response confi rms the effi ciency of SPA-Cys as an intermediate component for the creation of the immunosensor bioselective elements

In addition to affinity chromatography, the use of SPA, fused with a tag (enzymes, fluorescent proteins) is promising for diagnostics. As a fusion partner for the SPA was selected BAPmut. SPA-BAPmut was expressed in a soluble form in E. coli system. The target protein was obtained with purity more than 95 % using metal affinity chromatography. SPA-BAPmut is thermostable, and both parts of fusion protein (SPA and BAPmut) retain their IgG binding and alkaline phosphatase activity for a long time. As little as 0,2 ng of the antigen could be detected in Western blotting. The possibility of using SPA-BAPmut as universal secondary immunoreagent for different types of immunoassays was shown.

Number of publications: 10 articles (9 - in English-language journals), 10 abstracts. The total number of references to the author's publications / h-index of work according to databases is respectively: Web of Science - 12/2, Scopus - 21/3, Google Scholar - 81/6. One patent of Ukraine for an invention and one for a utility model of Ukraine were obtained.