%0 Journal Article
%T Construction, expression, functional §ãharacterization and practical application of fusion protein SPA-§£APmut
%A Gorbatiuk O. B.
%A Okunev O. V.
%A Nikolaev Yu. S.
%A Svyatenko O. V.
%J Biopolymers and Cell
%D 2013
%I Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
%R 10.7124/bc.000805
%X 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-§£APmut (> 1 g/l) were determined. The target protein was obtained with purity more than 95 % using  §®§¡§· method. SPA-§£APmut 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-§£APmut as universal secondary immunoreagent for different types of immunoassays was shown.
%K protein A
%K bacterial alkaline phosphatase
%K fusion protein
%K immunoassays
%U http://biopolymers.org.ua/pdf/uk/29/1/049/