Biopolym. Cell. 2013; 29(1):49-54.
http://dx.doi.org/10.7124/bc.000805
Molecular and Cell Biotechnologies
Construction, expression, functional characterization and practical application of fusion protein SPA-BAPmut
1Gorbatiuk O. B., 1, 2Okunev O. V., 1Nikolaev Yu. S., 1, 3Svyatenko O. V., 1, 2Kordium V. A.
  1. State Organization of Genetic and Regenerative Medicine NAMS Ukraine
    67, Vyshhorodska Str., Kyiv, Ukraine, 04114
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  3. National Aviation University
    Komarova, 1, Kyiv, Ukraine, 03058

Abstract

Aim. The creation of genetically engineered fusion protein SPA-BAPmut and its application as a secondary immunoreagent in immunoassays. Methods. Gene cloning, PCR, electrophoresis, DNA sequencing, bacteria cells culturing, protein expression and purification, ELISA, Western-blotting were used. Results. The DNA sequences encoding Staphylococcus aureus protein A (SPA) and bacterial alkaline phosphatase with enhanced catalytic activity (BAPmut) were used for construction of gene encoding fusion protein SPA-BAPmut that was expressed in the high-productive Escherichia coli system and obtained in a soluble form. Cultivation conditions to provide a high-level expression of SPA-BAPmut (> 1 g/l) were determined. The target protein was obtained with purity more than 95 % using IMAX method. 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. SPA-BAPmut was used as a substitute of secondary antibodies in immunoassays. As little as 5 ng of the antigen could be detected in Western blotting and 1 g/ml of IgG in ELISA. Conclusions. The possibility of using SPA-BAPmut as universal secondary immunoreagent for different types of immunoassays was shown.
Keywords: protein A, bacterial alkaline phosphatase, fusion protein, immunoassays
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