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ISSN 1995-5537

1 2012

"Biotechnology" journal V. 5, No.16, 2012
Р. 62-69, Bibliography 23, Russiuan.
Universal Decimal classification: 578.245:57.083.1:579.243:519.24

IN Escherichia coli BACTERIA

L. M. Korshun, I. V. Orlovskaya, L. M. Moysa, M. I. Vudmaska, A. I. Melnik,
A. Yu. Gorlov, S. V. Mikhalap, M. Ya. Spivak

JSC «Diaproph-Med», Kyiv
Zabolotny Institute of Microbiology and Virology of National Academy of Sciences of Ukraine, Kyiv

The scientific data received to the present time suggest about an important regulatory function of interferon in maintaining homeostasis, the presence of forward and backward connection between interferon, immune and neuroendocrinous systems, which generally form the basis of biological defense.

The use of interferon in the treatment of some diseases is based mainly on its antiviral, antiproliferative, immunomodulatory and radioprotective properties. However, the widespread introduction of these drugs, obtained from peripheral blood leukocytes of donors is limited by their high cost and lack of biologically safe materials. An important achievement in the study of the biological properties of human leukocyte interferon was the determination of its amino acid sequence. Using DNA technology has allowed to obtain the first artificial analogs of interferon suitable for use in clinical practice.

Cells of different microorganisms are widely used in modern biotechnological production of recombinant proteins. Purpose of the work was to select optimal conditions for induction of the biosynthesis of the soluble form of interferon alpha-2b human in Escherichia coli cells and development an effective technology for production of purified substance, suitable for preparations of interferon therapy appointment.

By using of a bacterial expression system based on plasmid vector pET32a in Escherichia coli strain Origami (DE3) cells a recombinant analogue of the natural leukocyte human interferon alfa-2b was obtained. The optimal cultivation conditions for the maximum yield of soluble forms of a target protein were chosen.

The three-step chromatographic purification scheme of recombinant alpha-2b interferon was developed, including cation- and anion-exchange chromatography with sorbents СM-toyopearl and DEAE-toyopearl, and gel filtration. The degree of purity of the obtained substance interferon alfa-2b was 96–98% and the yield of protein was 2.31 mg per 1 liter of medium.

Key words: recombinant human alpha-2b interferon, Escherichia coli bacteria, cultivation of strains, chromatographic purification of proteins.

© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2008