OBTAINING OF ScFv-CBD FUSION PROTEIN AND ITS APPLICATION FOR AFFINITY
PURIFICATION OF RECOMBINANT HUMAN INTERFERON ?2b
1Taras Shevchenko National University, Kyiv, Ukraine;
2Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv
The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon ?2b (IFN-?2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-?2b is shown.
Key words: single-chain antibodies, cellulose-binding domain, fusion proteins, bacterial inclusion bodies, protein refolding, protein immobilization, affinity purification.
Published at the site: 2006-09-14
The original article in Ukrainian is available for download in PDF format.
© The Ukrainian Biochemical Journal